Agonists can discriminate between cloned human and mouse prostacyclin receptors

The ability of prostacyclin analogues to stimulate adenylyl cyclase (AC) and phospholipase C (PLC) in Chinese hamster ovary (CHO) cells expressing cloned human (hIP) or cloned mouse (mIP) prostacyclin receptors has been compared. For hIP, the order of potency (pEC(50)) for stimulating AC and PLC pathways was similar: AFP-07 (9.3, 8.4)>cicaprost (8.3, 6.9), iloprost (7.9, 6.8)>taprostene (7.4, 6.8)>carbacyclin (6.9, 6.6), PGE(1) (6.6, 5.1). Although the standard IP agonists cicaprost and iloprost behaved similarly in both hIP and mIP receptor-expressing cells, carbacyclin and PGE(1) showed significantly higher potency at the mIP receptor, suggesting that the agonist recognition sites on hIP and mIP receptors are not identical. A further distinction between hIP and mIP receptors was found with taprostene, which had greater efficacy at hIP receptors (AC 94%, PLC 14%) than at mIP receptors (AC 77%, PLC 0%) (cicaprost=100% in each assay).     

Activation of IP prostanoid receptors prevents cardiomyocyte hypertrophy via cAMP-dependent signaling

The antihypertrophic action of angiotensin-converting enzyme inhibitors in the heart results partly from local potentiation of bradykinin. We have demonstrated that the antihypertrophic action of bradykinin is mediated by the release of nitric oxide from endothelium and elevation of cardiomyocyte cGMP. Whether other paracrine factors derived from the coronary endothelium, such as prostacyclin (PGI2), may act to prevent hypertrophy has not been explored. In the vasculature, activation by PGI2 of IP and EP1 prostanoid receptors elicits vasodilatation (via cAMP-dependent signaling) and vasoconstriction, respectively. The present objective was to determine whether IP prostanoid receptor activation has antihypertrophic actions in adult rat cardiomyocytes (ARCM). The selective IP agonist cicaprost (1 microM) virtually abolished the increase in [3H]phenylalanine incorporation (a marker of hypertrophy) induced either by endothelin-1 (ET-1; 60 nM, n = 10, P < 0.005) or by angiotensin II (1 microM, n = 6, P < 0.005). Cicaprost also inhibited ET-1 induction of c-fos mRNA expression, an additional marker of hypertrophy in ARCM (n = 5, P < 0.005). In the absence of hypertrophic stimuli, cicaprost alone did not significantly influence either marker. The antihypertrophic actions of cicaprost were mimicked by the dual IP/EP1 agonist iloprost (1 microM) in the presence of the EP1 antagonist AH-6809 (3 microM). Furthermore, cicaprost modestly but significantly increased cardiomyocyte cAMP content by 13 +/- 6% (P < 0.05, n = 4), and the antihypertrophic effect of cicaprost was lost in the presence of the cAMP-dependent protein kinase inhibitor H-89 (1 microM, n = 5, P < 0.05). However, ET-1 also induced increases in the activity of the intracellular growth signals ERK1 (by 3-fold) and ERK2 (by 5-fold) in ARCM, and these were not inhibited by cicaprost (P < 0.01, n = 5). Activation of IP receptors thus represents a novel approach to prevention of hypertrophy, and this effect is linked to cAMP-dependent signaling.     

Characterization of the PGI2/IP system in cultured rat mesangial cells

Mesangial cells play an important role in glomerular function. They are an important source of cyclooxygenase (COX)-derived arachidonic acid metabolites, including prostaglandin E(2) and prostacyclin. Prostacyclin receptor (IP) mRNA was amplified from cultured mesangial cell total RNA by RT-PCR. While the prostaglandin E(2) receptor subtype EP(2) was not detected, EP(1,3,4) mRNA was amplified. Also, IP protein was noted in mesangial cells, proximal tubules, inner medullary collecting ducts, and the inner and outer medulla. But no protein was detected in whole cortex preparations. Prostacyclin analogues: cicaprost and iloprost, increased cAMP levels in mesangial cells. On the other hand, arginine-vasopressin and angiotensin II increased intracellular calcium in mesangial cells, but cicaprost, iloprost and prostaglandin E(2) had no effect. Moreover, a 50% inhibition of cicaprost- and iloprost-cAMP stimulation was observed upon mesangial cell exposure to 25 and 35 mM glucose for 5 days. But no change in IP mRNA was observed at any glucose concentration or time exposure. Although 25 mM glucose had no effect on COX-1 protein levels, COX-2 was increased up to 50%. In contrast, PGIS levels were reduced by 50%. Thus, we conclude that the prostacyclin/IP system is present in cultured rat mesangial cells, coupling to a cAMP stimulatory pathway. High glucose altered both enzymes in the PGI(2) synthesis pathway, increasing COX-2 but reducing PGIS. In addition, glucose diminished the cAMP response to prostacyclin analogues. Therefore, glucose attenuates the PGI(2)/IP system in cultured rat mesangial cells.     

Factors affecting prostacyclin receptor agonist efficacy in different cell types

Octimibate and related nonprostanoid prostacyclin mimetics are partial agonists displaying highly tissue-specific responses. Octimibate demonstrated considerably greater efficacy for stimulation of adenylyl cyclase activity in Chinese hamster ovary cells transiently expressing mouse prostacyclin receptors (mIP-CHO cells) when compared to human SK-N-SH neuroblastoma cells, which endogenously express prostacyclin (IP) receptors. Pretreatment of both cell types with pertussis toxin (PTx) failed to influence IP agonist efficacy or potency, indicating a lack of involvement of an agonist-stimulated inhibitory G(i)-coupled pathway. Although stimulation of mIP-CHO cells with the full agonist cicaprost increased both [3H]cyclic AMP and [3H]inositol phosphate ([3H]IP) accumulation (pEC(50) values of 8.35 and 6.82, respectively), IP receptor signalling through G(q) in SK-N-SH cells was absent. Inhibition of protein kinase C (PKC) in mIP-CHO cells increased [3H]IP accumulation but had no effect on [3H]cyclic AMP accumulation. Therefore, the poor coupling of the IP receptor in SK-N-SH cells to G(q) is unlikely to explain the relatively low efficacy of octimibate for stimulating adenylyl cyclase in these cells. Furthermore, protein kinase A (PKA) inhibition appears to enhance IP receptor signalling through both G(s) and G(q) in mIP-CHO cells.     

Mechanism of cicaprost-induced desensitization in rat pulmonary artery smooth muscle cells involves a PKA-mediated inhibition of adenylyl cyclase

Long-term infusion of prostacyclin, or its analogs, is an effective treatment for severe pulmonary arterial hypertension. However, dose escalation is often required to maintain efficacy. The aim of this study was to investigate the mechanisms of prostacyclin receptor desensitization using the prostacyclin analog cicaprost in rat pulmonary artery smooth muscle cells (PASMCs). Desensitization of the cAMP response occurred in 63 nM cicaprost after a 6-h preincubation with agonist. This desensitization was reversed 12 h after agonist removal, and resensitization was inhibited by 10 microg/ml of cycloheximide. Desensitization was heterologous since desensitization to other G(s)alpha-adenylyl cyclase (AC)-coupled agonists, isoproterenol (1 microM), adrenomedullin (100 nM), or bradykinin (1 microM), was also reduced by preincubation with cicaprost. The reduced cAMP response to prolonged cicaprost exposure appeared to be due to inhibition of AC activity since the responses to the directly acting AC agonist forskolin (3 microM) and the selective AC5 activator NKH-477 were similarly reduced. Expression of AC2 and AC5/6 protein levels transiently decreased after 1 h of cicaprost exposure. The PKA inhibitor H-89 (1 microM) added 1 h before cicaprost preincubation (6 h, 63 nM) completely reversed cicaprost-induced desensitization, whereas the PKC inhibitor bisindolylmaleimide (100 nM) was only partly effective. Desensitization was not prevented by the G(i) inhibitor pertussis toxin. In conclusion, chronic treatment of PASMCs with cicaprost induced heterologous, reversible desensitization by inhibition of AC activity. Our data suggest that heterologous G(s)alpha desensitization by cicaprost is mediated predominantly by a PKA-inhibitable isoform of AC, most likely AC5/6.     

The effect of prostacyclin agonists on the differentiation of phorbol ester treated human erythroleukemia cells

Phorbol-12-myristate-13-acetate (PMA) induces megakaryocytopoeisis in human erythroleukemia (HEL) cells which is characterized by the increase in cell size, increase in nuclear polyploidization and expression of megakaryocyte marker, CD41. However, upon treatment with 100 nM of selective prostacyclin (IP) agonist beraprost inhibits the induced differentiation. Moreover, selective non-prostanoid IP agonist, BMY 45778 prevents PMA induced megakaryocytopoeisis in HEL cells similarly, while prostaglandin E(2) and specific EP(3) agonist sulprostone have no effect. Thus, IP receptor is involved. Furthermore, adenylate cyclase activator forskolin and cAMP analog dibutyryl-cAMP also prevented PMA induced megakaryocytopoeisis in HEL cells. Thus, IP agonists inhibition of PMA induced megakaryocytopoeisis in HEL cells may involve a cAMP dependent pathway.     

The alpha, but not the beta, isoform of the human thromboxane A2 receptor is a target for prostacyclin-mediated desensitization

In this study, we examined the effects the prostacyclin receptor (IP) agonist cicaprost exhibited on U46619-mediated thromboxane A(2) receptor (TP) signaling in platelets and compared it to that which occurs in human embryonic kidney (HEK) 293 cells stably overexpressing the individual TPalpha or TPbeta isoforms. Consistent with previous studies, cicaprost abrogated U46619-mediated platelet aggregation and mobilization of intracellular calcium ([Ca(2+)](i)). In HEK 293 cells, signaling by TPalpha, but not TPbeta, was subject to IP-mediated desensitization in a protein kinase A-dependent, protein kinase C-independent manner. Desensitization of TPalpha signaling was independent of the nature of the IP agonist used, the level of IP expression, or the subtype of G(q) protein. Signaling by TP(Delta)(328), a truncated variant of TP devoid of the divergent residues of the TPs, or by TPalpha(S329A), a site-directed mutant of TPalpha, were insensitive to IP agonist activation. Whole cell phosphorylations established that TPalpha, but not TPbeta or TPalpha(S329A), is subject to IP-mediated phosphorylation and that TPalpha phosphorylation is inhibited by H-89. Thus, we conclude that TPalpha, but not TPbeta, is subject to cross-desensitization by IP mediated through direct protein kinase A phosphorylation at Ser(329) and propose that TPalpha may be the isoform physiologically relevant to TP:IP-mediated vascular hemostasis.     

Properties of chimeric prostacyclin/prostaglandin D2 receptors: site-directed mutagenesis reveals the significance of the isoleucine residue at position 323

Mouse prostacyclin (mIP) receptors transiently expressed in Chinese hamster ovary (CHO) cells activated both adenylyl cyclase and phospholipase C, with a 33-fold preference for signaling through Gs. The prostacyclin (IP) receptor agonists cicaprost, iloprost, carbacyclin, and prostaglandin E1 showed a similar order of potency for activation of both signaling pathways in cells transiently transfected with the mIP and the chimeric prostacyclin/prostaglandin D2 (IPN-VII/DPC and IPN-V/DPVI-C) receptors. Substitution of the carboxyl-terminal tail of the prostacyclin receptor with the corresponding region of the mDP receptor (IPN-VII/DPC) produced a receptor with increased coupling to both Gs and Gq. However, this increased G-protein coupling was lost in the IPN-V/DPVI-C receptor. The observation that both these chimeric receptors can activate phospholipase C indicates that the carboxyl-terminal tail of the IP receptor is not entirely responsible for its ability to couple to Gq. Site-directed mutagenesis studies suggest that isoleucine at position 323 in the IPN-VII/DPC receptor plays an important role in mediating the increased potency of this chimeric receptor.     

Prostaglandin production by the pregnant and non-pregnant rabbit uterus

We have studied the prostaglandin synthesis of the pregnant and non-pregnant rabbit uterus in a microsomal membrane preparation, and in an ex vivo perfused uterus preparation which retains agonist stimulated prostaglandin production. In both the microsomal and isolated perfused system, prostacyclin was the major arachidonic acid metabolite produced; PGE2 was also produced in substantial quantities while TxB2 and PGF2 alpha were not detectable. Moreover, oxytocin was a specific stimulus of PGE2 release. The steroid hormone milieu influenced the level of agonist stimulated prostaglandin release; in general, ovariectomized, estrogen treated animals were more responsive to agonist stimulation than those treated with estrogen followed by progesterone. The microsomal studies indicated that the pregnant animal had a greatly enhanced capacity to metabolize arachidonic acid when compared with the non-pregnant animal. However, this was not reflected in the ability of agonists to stimulate prostaglandin release in the ex vivo perfused preparation.     

The PPARβ/δAgonist GW0742 Relaxes Pulmonary Vessels and Limits Right Heart Hypertrophy in Rats with Hypoxia-Induced Pulmonary Hypertension

Background

Pulmonary vascular diseases are increasingly recognised as important clinical conditions. Pulmonary hypertension associated with a range of aetiologies is difficult to treat and associated with progressive morbidity and mortality. Current therapies for pulmonary hypertension include phosphodiesterase type 5 inhibitors, endothelin receptor antagonists, or prostacyclin mimetics. However, none of these provide a cure and the clinical benefits of these drugs individually decline over time. There is, therefore, an urgent need to identify new treatment strategies for pulmonary hypertension.

Methodology/Principal Findings

Here we show that the PPARβ/δ agonist GW0742 induces vasorelaxation in systemic and pulmonary vessels. Using tissue from genetically modified mice, we show that the dilator effects of GW0742 are independent of the target receptor PPARβ/δ or cell surface prostacyclin (IP) receptors. In aortic tissue, vascular relaxant effects of GW0742 were not associated with increases in cGMP, cAMP or hyperpolarisation, but were attributed to inhibition of RhoA activity. In a rat model of hypoxia-induced pulmonary hypertension, daily oral dosing of animals with GW0742 (30 mg/kg) for 3 weeks significantly reduced the associated right heart hypertrophy and right ventricular systolic pressure. GW0742 had no effect on vascular remodelling induced by hypoxia in this model.

Conclusions/Significance

These observations are the first to show a therapeutic benefit of ‘PPARβ/δ’ agonists in experimental pulmonary arterial hypertension and provide pre-clinical evidence to favour clinical trials in man.     

Classification of kinin receptors

This minireview is divided into three parts: the first part refers to the characterization and classification of kinin receptors using agonists and antagonists in isolated tissues (classical pharmacology). Two kinin receptors have been considered on the basis of their distinct pharmacology, namely the B1 receptor of the rabbit aorta (rank order of potency of agonists: LysdesArg9BK > desArg9BK > or = LysBK > BK; apparent affinities of antagonists Lys[Leu8]desArg9BK (pIC50 8.4) > [Leu8]desArg9BK (pIC50 7.4) > HOE 140, a B2 receptor antagonist, pIC50<5.0), and the B2 receptor of the rabbit jugular vein (potency of agonists: LysBK = BK > LysdesArg9BK = desArg9BK and HOE 140 (pIC50 9.0) > Lys[Leu8]desArg9BK, pIC50<5.0). The second part describes species-related B1 receptor subtypes, demonstrated by different pharmacological profiles of agonists and antagonists: human, rabbit and pig subtypes (LysdesArg9BK > desArg9BK and Lys[Leu8]desArg9BK > [Leu8]desArg9BK) and dog, rat, mouse and hamster B1 receptors (desArg9BK = LysdesArg9BK and [Leus]desArg9BK = Lys[Leu8]desArg9BK). Affinities of agonists and antagonists in some species (man, rabbit, pig) are significantly increased (at least 10-fold) by the presence of a Lys at their N-terminus. The last part describes species-related B2 receptor subtypes supported by results obtained with non-peptide receptor agonists (FR 190997) and antagonists (FR 173657). While BK acts as a full agonist in man, rabbit and pig, FR 190997 behaves as a full agonist on human, as partial agonist on rabbit, and as pure antagonist on pig B2 receptors. Various hypotheses are considered to interpret these findings.     

Characterization of bradykinin receptors in peripheral organs.

Bradykinin (BK) and related kinins are potent stimulants of the rabbit jugular vein, the hamster urinary bladder, and the guinea pig trachea. The characterization of kinin receptors in these tissues was made with agonists and antagonists. Results obtained with agonists indicate that bradykinin and kallidin are much more active than des-Arg9-BK and suggest the presence of B2 receptors in the three organs. Some new agonists were also tested and the BK analogue, [Hyp3,Tyr(Me)8]BK, was found to be a potent and selective stimulant of the three preparations, with pD2 values of 8.56, 8.00, and 8.39, respectively, but inactive on the rabbit aorta (a B1-receptor system). Contractile effects of kinins in the rabbit jugular vein and hamster urinary bladder were reduced or eliminated by B2-receptor antagonists but at different concentration levels; e.g., acetyl-D-Arg[Hyp3,D-Phe7]BK showed pA2 values of 7.78 on the rabbit jugular vein but only 5.72 on hamster urinary bladder. This compound contracted the guinea-pig trachea and was found to be inactive as an antagonist on this preparation. Contractions of the hamster urinary bladder and the guinea-pig trachea in response to bradykinin were markedly reduced or eliminated by indomethacin and by BW 755C, while those of the rabbit jugular vein were not modified. The present findings indicate that the myotropic effect of kinins on the rabbit jugular vein depends on the activation of B2 receptors and suggest that B2 receptors are largely responsible also for the response of the hamster urinary bladder. B2 receptors and (or) a nonreceptor mechanism appear to be involved in the stimulant effects of the kinin agonists and some antagonists in the guinea-pig trachea.     

Iloprost-induced desensitization of the prostacyclin receptor in isolated rabbit lungs

Background

The rapid desensitization of the human prostacyclin (IP) in response to agonist binding has been shown in cell culture. Phosphorylation of the IP receptor by protein kinase C (PKC) has been suggested to be involved in this process.

Methods and results

In this study we investigated the vasodilatory effects of iloprost, a stable prostacyclin analogue, in perfused rabbit lungs. Continuous infusion of the thromboxane mimetic U46619 was employed to establish stable pulmonary hypertension. A complete loss of the vasodilatory response to iloprost was observed in experiments with continuous iloprost perfusion, maintaining the intravascular concentration of this prostanoid over a 180 min period. When lungs under chronic iloprost infusion were acutely challenged with inhaled iloprost, a corresponding complete loss of vasoreactivity was observed. This desensitization was not dependent on upregulation of cAMP-specific phosphodiesterases or changes in adenylate cyclase activity, as suggested by unaltered dose-response curves to agents directly affecting these enzymes. Application of a prostaglandin E1 receptor antagonist 6-isopropoxy-9-oxoxanthene-2-carboxylic acid (AH 6809) or the PKC inhibitor bisindolylmaleimide I (BIM) enhanced the vasodilatory response to infused iloprost and partially prevented tachyphylaxis.

Conclusion

A three-hour infusion of iloprost in pulmonary hypertensive rabbit lungs results in complete loss of the lung vasodilatory response to this prostanoid. This rapid desensitization is apparently not linked to changes in adenylate cyclase and phosphodiesterase activation, but may involve PKC function and co-stimulation of the EP1 receptor in addition to the IP receptor by this prostacyclin analogue.     

Aqueous flare elevation induced by transcorneal application of highly selective agonists for prostaglandin E2 receptor subtypes in pigmented rabbits: effect of tetramethylpyrazine.

We examined the disruptive effect of highly selective agonists for prostaglandin E2 receptor subtypes (EP1, EP2, EP3 and EP4) on the blood-aqueous barrier, and evaluated the inhibitory effect of tetramethylpyrazine, an active component of Ligusticum wallichii, on the elevation of aqueous flare induced by the EP agonists in pigmented rabbits. Highly selective EP agonists (ONO-DI-004, EP1 agonist; ONO-AE1-259-01, EP2 agonist; ONO-AE-248, EP3 agonist; ONO-AE1-329, EP4 agonist) at 12.5 to 250 microg/ml were transcorneally administered to the eyes of pigmented rabbits using a glass cylinder. Animals were pretreated intravenously with tetramethylpyrazine (10 or 30 mg/kg) 30 minutes before application of the EP2 or the EP4 agonist. Aqueous flare was measured using a laser flare-cell meter. Aqueous flare intensity was expressed as the area under the curve (AUC) in arbitrary units. After administration of ONO-AE1-259-01 or ONO-AE1-329, aqueous flare increased and then gradually decreased. ONO-DI-004 and ONO-AE-248 had almost no effect on aqueous flare elevation. The AUC of eyes in rabbits pretreated with tetramethylpyrazine, 10 or 30 mg/kg i.v., was significantly smaller than that of eyes in rabbits treated with ONO-AEI-259-01 alone. The AUC of eyes in rabbits pretreated with tetramethylpyrazine, 10 or 30 mg/kg i.v., was not significantly smaller than that of eyes in rabbits treated with ONO-AEI-329 only. The results indicated that EP2 and EP4 agonists induced aqueous flare elevation in pigmented rabbits, and that tetramethylpyrazine inhibited the aqueous flare elevation induced by the EP2 agonist but did not suppress the elevation induced by the EP4 agonist.     

Prostaglandin production by the pregnant and non-pregnant rabbit uterus

We have studied the prostaglandin synthesis of the pregnant and non-pregnant rabbit uterus in a microsomal membrane preparation, and in an perfused uterus preparation which retains agonist stimulated prostaglandin production. In both the microsomal and isolated perfused system, prostacyclin was the major arachidonic acid metabolite produced; PGE₂ was also produced in substantial quantities while TxB₂ and PGF_2α were not detectable. Moreover, oxytocin was a specific stimulus of PGE₂ release. the steroid hormone milieu influenced the level of agonist stimulated prostaglandin release; in general, ovariectomized, estrogen treated animals were more responsive to agonist stimulation than those treated with estrogen followed by progesterone. The microsomal studies indicated that the pregnant animal had a greatly enhanced capacity to metabolize arachidonic acid when compared with the non-pregnant animal. However, this was not reflected in the ability of agonists to stimulate prostaglandin release in the perfused preparation.     

Modulation of cytokine release from mononuclear cells by prostacyclin, IL-4 and IL-13

In a previous study, we reported that cicaprost, a stable prostacyclin analogue can inhibit the release of granulocyte-macrophage colony-stimulating factor (GM-CSF) from activated human peripheral mononuclear blood cells (PBMCs). Since interleukin (IL-4) and IL-13 have been shown to inhibit the release of cytokines from PBMCs we tested the hypothesis that prostacyclin in combination with IL-4 or IL-13 can act synergistically to modulate the release of IL-10, generally associated with anti-inflammatory properties, and the pro-inflammatory cytokine tumour necrosis factor alpha (TNF-alpha). For this purpose, PBMCs were isolated over Ficoll, stimulated with lipopolysaccharide (LPS) and incubated in the presence of cicaprost, IL-4 or IL-13. There was a significant reduction in TNF-alpha as well as IL-10 secretion from LPS-stimulated PBMCs following incubation with IL-4 or IL-13. In contrast, cicaprost reduced the secretion of TNF-alpha but led to a slight enhancement of IL-10 release from PBMCs. When LPS-activated PBMCs were incubated in the presence of cicaprost and IL-4 or IL-13 there was a selective, synergistic inhibition of the TNF-alpha release which was not observed for IL-10. Thus, our data suggest that prostacyclin can synergize with cytokines to selectively inhibit the release of pro-inflammatory cytokines from PBMCs.     

Role of prostaglandin E2 receptor subtypes in ovarian follicle growth in the rat in vivo. Correlation with interleukin-8 and neutrophils

This study was conducted to elucidate the role of three of prostaglandin E2 (PGE2) receptor subtype (EP2, EP3, and EP4) agonists in the process of follicular growth. The influence of these agonists on ovarian expression of intimately related factors to follicle development (neutrophils and interleukin-8 (IL-8)) was also investigated. Immature female Wistar rats were injected once with these agonists and killed 48 hours later. Another group of rats were injected pregnant mare serum gonadotrophin. For evaluation of follicle growth, morphometric assessment of antral and ovulatory follicles was performed in serial ovarian sections. The study demonstrated that, EP2 and EP4 agonists showed the maximum follicle counts and diameters versus the control. EP2 and EP4 agonists mimicked PMSG induced follicle growth. Injection of the three agonists induced neutrophil infiltration into theca layer. EP4 agonist showed the most intense ovarian neutrophil accumulation. In addition, dense ovarian IL-8 expression was observed only after EP4 agonist injection. CONCLUSIONS: Our data suggests that: 1) EP2 and EP4 receptors are the key PGE2 receptors engaged in follicle growth. 2) Ovarian IL-8 expression and neutrophil infiltration are chiefly mediated via the EP4 receptor. EP2 and EP4 receptor agonists may be candidates for promising reagents that induce follicle maturation in clinical or agricultural fields. This knowledge could provide numerous targets for manipulation of fertility.     

Interaction of the human prostacyclin receptor with the PDZ adapter protein PDZK1: role in endothelial cell migration and angiogenesis

Prostacyclin is increasingly implicated in re-endothelialization and angiogenesis but through largely unknown mechanisms. Herein the high-density lipoprotein (HDL) scavenger receptor class B, type 1 (SR-B1) adapter protein PDZ domain-containing protein 1 (PDZK1) was identified as an interactant of the human prostacyclin receptor (hIP) involving a Class I PDZ ligand at its carboxyl terminus and PDZ domains 1, 3, and 4 of PDZK1. Although the interaction is constitutive, it may be dynamically regulated following cicaprost activation of the hIP through a mechanism involving cAMP-dependent protein kinase (PK)A-phosphorylation of PDZK1 at Ser-505. Although PDZK1 did not increase overall levels of the hIP, it increased its functional expression at the cell surface, enhancing ligand binding and cicaprost-induced cAMP generation. Consistent with its role in re-endothelialization and angiogenesis, cicaprost activation of the hIP increased endothelial cell migration and tube formation/in vitro angiogenesis, effects completely abrogated by the specific IP antagonist RO1138452. Furthermore, similar to HDL/SR-B1, small interfering RNA (siRNA)-targeted disruption of PDZK1 abolished cicaprost-mediated endothelial responses but did not affect VEGF responses. Considering the essential role played by prostacyclin throughout the cardiovascular system, identification of PDZK1 as a functional interactant of the hIP sheds significant mechanistic insights into the protective roles of these key players, and potentially HDL/SR-B1, within the vascular endothelium.