Prostanoids receptors signaling in different diseases/cancers progression

Abstract

Prostanoids, that is, prostaglandins (PGs) PGE₂, PGF_2α, PGI₂, PGD₂ and thromboxane A₂(TXA₂), are the oldest members of the eicosanoid family. The PGs are a family of lipid mediators formed in response to various stimuli. They are transported into the extracellular microenvironment by specific multidrug resistance-associated proteins (MRPs) after synthesis. Once exported to the microenvironment, prostanoids bind to G-protein coupled receptors that contain seven transmembrane spanning domains. There are eight types of the prostanoid receptors conserved in mammals from mouse to human. They are the PGD receptor (DP), four subtypes of the PGE receptor (EP₁, EP₂, EP₃ and EP₄), the PGF receptor (FP), PGI receptor (IP) and TXA receptor (TP). Recently, several studies have revealed the roles of PG receptor signaling in various pathological conditions, and suggest that selective manipulation of the prostanoid receptors may be beneficial in treatment of the pathological conditions. Here we review these recent findings of roles of prostanoid receptor signaling and their therapeutic implications.     

Lipidomic analysis reveals prostanoid profiles in human term pregnant myometrium

Prostanoids modulate the activity of human pregnant myometrium and their functional role can be appreciated through characterisation of prostanoid receptors and tissue concentration of prostanoids. We have applied a lipidomic approach to elucidate the profile of prostanoids in human non-labouring and labouring myometrium. We have identified a total of nineteen prostanoids including prostacyclin, thromboxanes, prostaglandins and dihydro-prostaglandins. Prostacyclin was the predominant prostanoid in both non-labouring and labouring myometria, with PGD₂ and PGF_2α being the second most abundant. Although the total amount of prostanoids was increased in the labouring tissue, PGE₂ and 13,14-dihydro-15-keto-PGE₂ were the only prostanoids to increase significantly at early and late labour (p≤0.001). Our data suggest that PGF_2α plays an important role in parturition, whilst the increase in PGE₂ could occur to facilitate cervical dilation and relaxation of the lower myometrium during labour. Although the elevation in TXA₂ was less marked than expected, in terms of translation to function even a relatively small increase in the level of this potent spasmogen may have significant effects.     

Studies of the characterisation of prostanoid receptors: A proposed classification

Comparison of rank orders of agonist potency of the anturally occurring prostanoids. PGD₂, PGE₂, PGF₂α and PGI₂ as well as the stable TxA₂ mimetic, U-46619, on a range of smooth muscle preparations provides evidences evidence for the existence of distinct receptors for PGE₂. PGF₂α and TxA₂. Since others have provided evidence for the existence of distinct receptors for PGD₂ and PGI₂, we suggest that receptors exist for each of these naturally occurring 2-series prostanoids. Results obtained with two specific prostanoid receptor blocking drugs, SC-19220 and AH 19437, supported and extend these conclusions. SC-19220 selectively block some but not all PGE-sensitive receptors. While AH 19437 selectively blocks all U-46619/TxA₂-sensitive receptors. A nomenclature for prostanoid receptors is proposed,; in which each receptor is designated the letter P preceded by a letter signifying the most potent natural prostanoid agonist at than receptor, such that receptors sensitivity to PGs D₂, E₂, F₂α, I₂ and TxA₂ become DP-, EP-, FP- and TP- receptors respectively. Where some sub-division is required within a receptor group, e.g. EP-receptors (SC-19220-sensitive and SC-19220-insensitive), subcript numbers may be used such that these are EP₁ and EP₂ subtypes. The resulting scheme is a working hypothesis and its confirmation requires the development of potent selective prostanoid receptor blocking drugs for each postulated type.     

Immunological and non-immunological synthesis and release of prostaglandins and thromboxanes from isolated guinea pig trachea

Specific radioimmunoassays were used to demonstrate the synthesis by the guinea pig trachea of 6-keto PGF_1α, TxB₂, and PGF_2α in addition to PGE₂. The rank order of both spontaneous and stimulated release was PGE₂ > PGF_2α > 6-keto PGF_1α = TxB₂. Ovalbumin-induced prostanoid release from sensitized tissue was antigen-specific. The release was unlikely to be a secondary consequence of tracheal contraction since incubations with calcium ionophore A23187, at a concentration which produces an equivalent magnitude of contraction of sensitized trachea, did not induce a significant PG or Tx production. In contrast, significantly higher prostanoid synthesis was induced by A23187 in unsensitized than sensitized trachea. Thus sensitization altered the profile of arachidonic acid metabolism evoked by the ionophore.     

Cellular and molecular effects of unoprostone as a BK channel activator

Effects of unoprostone isopropyl (unoprostone), a prostaglandin metabolite analog; latanoprost, a PGF_2α analog; and PGF_2α were examined in HCN-1A cells, a model system for studies of large conductance Ca²⁺ activated K⁺(BK) channel activator-based neuroprotective agents. Unoprostone and latanoprost, both used as anti-glaucoma agents, have been suggested to act through FP receptors and have neuroprotective effects. Ion channel activation, plasma membrane polarization, [Ca²⁺]_i changes and protection against long-term irreversible glutamate-induced [Ca²⁺]_i increases were studied. Unoprostone activated iberiotoxin (IbTX)-sensitive BK channels in HCN-1A cells with an EC₅₀ of 0.6 ± 0.2 nM and had no effect on Cl⁻ currents. Unoprostone caused IbTX-sensitive plasma membrane hyperpolarization that was insensitive to AL8810, an FP receptor antagonist. In contrast, latanoprost and PGF_2α activated a Cl⁻ current sensitive to [Ca²⁺]_i chelation, tamoxifen and AL8810, and caused IbTX-insensitive, AL8810-sensitive membrane depolarization consistent with FP receptor-mediated Ca²⁺ signaling Cl⁻ current activation. Latanoprost and PGF_2α, but not unoprostone, increased [Ca²⁺]_i. Unoprostone, PGF_2α only partially, but not latanoprost protected HCN-1A cells against glutamate-induced Ca²⁺ deregulation. These findings show that unoprostone has a distinctly different mechanism of action from latanoprost and PGF_2α. Whether unoprostone affects the BK channel directly or an unidentified signaling mechanism has not been determined.     

Fine particulate matter (PM2.5) enhances FcεRI-mediated signaling and mast cell function

Persistent exposure to ambient fine particulate matter (PM_2.5) can exacerbate allergic diseases in humans. Mast cells play an important role in allergic inflammation in peripheral tissues, such as skin, mucosa, and lung. Engagement of the high-affinity Fc receptor leads to mast cell degranulation, releasing a variety of highly active mediators including histamine, leukotrienes, and inflammatory cytokines. How PM_2.5 exposure affects mast cell activation and function remains largely unknown. To characterize the effect of PM_2.5 on mast cells, we used bone marrow-derived mast cells (BMMCs) to examine whether PM_2.5 affected FcεRI-mediated signaling, cytokine production, and degranulation. Exposure to high doses of PM_2.5 caused pronounced apoptosis and death of BMMCs. In contrast, exposure to low doses of PM_2.5 enhanced mast cell degranulation and FcεRI-mediated cytokine production. Further analysis showed that PM_2.5 treatment increased Syk activation and subsequently phosphorylation of its substrates including LAT, PLC-γ1, and SLP-76. Moreover, PM_2.5 treatment led to activation of the PI3K and MAPK pathways. Intriguingly, water-soluble fraction of PM_2.5 were found responsible for the enhancement of FcεRI-mediated signaling, mast cell degranulation, and cytokine production. Our data suggest that PM_2.5, mainly water-soluble fraction of PM_2.5, could affect mast cell activation through enhancing FcεRI-mediated signaling.     

Production of prostaglandin F2α by molecular breeding of an oleaginous fungus Mortierella alpina

Cyclooxygenases are responsible for the production of prostaglandin H₂ (PGH₂) from arachidonic acid. PGH₂ can be converted into some bioactive prostaglandins, including prostaglandin F_2α (PGF_2α), a potent chemical messenger used as a biological regulator in the fields of obstetrics and gynecology. The chemical messenger PGF_2α has been industrially produced by chemical synthesis. To develop a biotechnological process, in which PGF_2α can be produced by a microorganism, we transformed an oleaginous fungus, Mortierella alpina 1S-4, rich in triacylglycerol consisting of arachidonic acid using a cyclooxygenase gene from a red alga, Gracilaria vermiculophylla. PGF_2α was accumulated not only in the mycelia of the transformants but also in the extracellular medium. After 12 days of cultivation approximately 860 ng/g and 6421 µg/L of PGF_2α were accumulated in mycelia and the extracellular medium, respectively. The results could facilitate the development of novel fermentative methods for the production of prostanoids using an oleaginous fungus.     

Anticonvulsive effects of endogenous prostaglandins formed in brain of spontaneously convulsing gerbils

Seizures were induced in a strain of epileptic gerbils by moderate environmental stress. Concentrations of five different cyclooxygenase products (PGD₂, PGF_2α, PGE₂, 6-keto-PGF_1α and thromboxane B₂) were measured in brain by specific radioimmunoassays before and at different time intervals after the onset of clonic-tonic convulsions. All prostanoids markedly increased subsequent to the convulsions. Maximal concentrations were reached after about 15 min. The major compound detected was PGD₂, followed by PGF_2α and lower concentrations of the other cyclooxygenase products. Indomethacin completely prevented the convulsion-induced formation of prostanoids. Fifteen min after a first seizure almost all animals proved to be protected against a second convulsion. Indomethacin pretreatment markedly reduced the number of convulsion-resistant animals. These findings are compatible with the hypothesis that endogenous prostaglandins exert anticonvulsive effects.     

Prostanoid synthesis in whole blood cells from fish of the Arabian Gulf

The ability to synthesise prostaglandins and thromboxane from ¹⁴C-labelled arachidonic acid was investigated in 11 species of fish from the Arabian Gulf. Cyclooxygenase activity was assessed in washed whole blood cells. Arachidonic acid and its metabolites were extracted and separated on silicic acid columns and thin layer chromatography (silica gel G). Total capacity to convert [¹⁴C]arachidonic acid to prostanoids varied from 1 to 35% among the 11 fish species studied. Gray shark (Chiloscyllium griseum) blood cells had the highest capacity (37±0.4%) to convert arachidonate into prostanoids and two species of catfish (Arius bilineatus and A. thalassinus) exhibited greater than 10% capacity to convert [¹⁴C]arachidonate into prostanoids. The major prostanoid synthesised by the two catfish (A. bilineatus and A thalassinus) was 6-keto PGF_1α, a stable metabolite of prostacyclin, PGI₂. In contrast, A. teunispinis synthesised thromboxane B₂, a stable metabolite of thromboxane A₂. Thromboxane B₂ (TXB₂) was the major product synthesised by all three species of shark studied (Chil. griseum, Carcharhinus plumbeus, Carch. melanopterus), with 6-keto PGF_1α a minor product. Other fish studied showed a varied pattern of prostanoid synthesis. The synthesis of these prostanoids was almost completely blocked by preincubation of the whole blood cells from catfish and shark with indomethacin (0.5 μM) suggesting the involvement of cyclooxygenase-mediated prostanoid synthesis.     

Induction of Prostanoid Biosynthesis by Bacterial Lipopolysaccharide and Isoproterenol in Rat Microglial Cultures

Abstract: We have used purified microglial cultures obtained from neonatal rat cerebral cortex to investigate the ability of microglia to release prostanoids after exposure to bacterial lipopolysaccharide, a classic macrophage activator. Release of prostaglandin E₂, prostaglandin D₂, and thromboxane A₂ was low in basal conditions and increased in a dose- and time-dependent way upon lipopolysaccharide treatment (1–100 ng/ml), by a mechanism requiring de novo protein synthesis. When compared with astrocytes, microglial cells appeared to respond more effectively to lipopolysaccharide, being able to release prostanoids after exposure to a 100-fold lower concentration of lipopolysaccharide. In addition to prostanoids, we also measured the release of leukotriene B₄; although lipopolysaccharide failed to stimulate leukotriene B₄ release by microglial cells, it doubled the basal production in astrocytes. Lipopolysaccharide enhanced the release of preloaded [³H]arachidonic acid from microglial membrane phospholipids by a mechanism inhibited by the protein synthesis inhibitor cycloheximide, which suggests that the increased availability of arachidonic acid contributed to the enhanced prostanoid production. Lipopolysaccharide, however, also stimulated prostanoid synthesis by inducing cyclooxygenase activity, as shown by determining the activity of newly synthesized enzyme after inactivating the endogenous enzyme with aspirin and by assessing the level of the inducible form of cyclooxygenase by western blot analysis. Among the mechanisms potentially involved in the regulation of microglial prostanoid production, we studied the effect of β-adrenergic receptor activation. The β-agonist isoproterenol was inactive by itself but doubled the effect of lipopolysaccharide. The drug appeared to act mainly through the inducible cyclooxygenase; because it did not stimulate arachidonic acid release, it enhanced the lipopolysaccharide-evoked prostanoid production observed after aspirin pretreatment and induced de novo synthesis of cyclooxygenase detectable by western blot analysis. We suggest that during cerebral inflammatory processes microglia can contribute to the establishment of high prostanoid levels, which can be further elevated by β-adrenergic activation.     

Comparison of prostanoid forming capacity of neuronal and astroglial cells in primary cultures

Prostaglandin (PG) and thromboxane (TX) biosynthesis in primary neuronal and astroglial cell cultures was studied. Cultures obtained from fetal (15–16 days old) and neonatal rat brain hemispheres were characterized by chemical and immunocytochemical staining techniques as predominantly neurons or mature and immature astrocytes, respectively. Six-day old neuronal cell cultures grown in the presence of cytosine arabinoside (2 μM) from the day 3 onwards were contaminated up to 10% with glioblasts. In astroglial cultures up to 3% of the cells were postively stained with a marker for oligodendroglial cells. Fibroblast contamination was below 1% in both cultures. Prostanoid formation (measured by specific radioimmunoassays) in 6-day old neuronal cell cultures was low (sum of the amount of PGs and TX formed: 1.16 ± 0.17 (ng/mg protein/15 min) as compared to 14-day old cultured astroglial cells: 21.27 ± 2.53 (ng/mg protein/15 min). Also the pattern of prostanoids formed was different in neuronal (PGD₂ ? PGF_2α > TXB₂ ? PGE₂) and astroglial cells (PGD₂ > TXB₂ ? PGF_2α ? PGE₂ ? 6-ketoPGF_1α). Preincubation with arachidonic acid (1 μg/ml) did not affect prostanoid formation in both cultures, whereas it was stimulated 4–6-fold by addition of the calcium ionophore A23187 (1 μM). These results, although found on cultured neuronal and glial cells of different stages of development, support the view that astroglial cells might play a crucial role in brain prostanoid synthesis.     

Inhibitory effect of hot-water extract of quince (<Emphasis Type="Italic">Cydonia oblonga</Emphasis>) on immunoglobulin E-dependent late-phase immune reactions of mast cells

We evaluated the effect of a crude hot-water extract (HW) of quince (Cydonia oblonga Miller) fruit on immunoglobulin E (IgE)-dependent late-phase immune reactions of mast cells using in vitro system. Mast cell-like RBL-2H3 cells were treated with quince HW and late-phase reaction was then induced by stimulation with IgE + Antigen. Quince HW reduced the elevation of interleukin-13 and tumor necrosis factor-α expression level. Furthermore, quince HW suppressed these cytokine expressions of mouse bone marrow-derived mast cells (BMMCs), a normal mast cell model. Leukotriene C₄ and prostaglandin D₂ production in BMMCs after 1 and 6 h of stimulation, respectively, were also reduced by treating the cells with quince HW. We found that the induction of intracellular cyclooxygenase (COX)-2 expression but not COX-1 expression in BMMCs was reduced by quince HW. These results suggest that quince HW has an inhibitory effect on broad range of the late-phase immune reactions of mast cells.     

The role of activated adenosine receptors in degranulation of human LAD2 mast cells

Mast cell degranulation triggers hypersensitivity reactions at the body–environment interface. Adenosine modulates degranulation, but enhancement and inhibition have both been reported. Which of four adenosine receptors (ARs) mediate modulation, and how, remains uncertain. Also uncertain is whether adenosine reaches mast cell ARs by autocrine ATP release and ecto-enzymatic conversion. Uncertainties partly reflect species and cell heterogeneity, circumvented here by focusing on homogeneous human LAD2 cells. Quantitative PCR detected expression of A_2A, A_2B, and A₃, but not A₁, ARs. Nonselective activation of ARs with increasing NECA monotonically enhanced immunologically or C3a-stimulated degranulation. NECA alone stimulated degranulation slightly. Selective AR antagonists did not affect C3a-stimulated degranulation. NECA''s enhancement of C3a-triggered degranulation was partially inhibited by separate application of each selective antagonist, and abolished by simultaneous addition of antagonists to the three ARs. Only the A_2A antagonist separately inhibited NECA''s enhancement of immunologically stimulated degranulation, which was abolished by simultaneous addition of the three selective antagonists. Immunological or C3a activation did not stimulate ATP release. NECA also enhanced immunologically triggered degranulation of mouse bone marrow derived mast cells (BMMCs), which was partially reduced only by simultaneous addition of the three antagonists or by the nonselective antagonist {"type":"entrez-protein","attrs":{"text":"CGS15943","term_id":"875345334"}}CGS15943. BMMCs also expressed A_2A, A_2B, and A₃ ARs. but not A₁AR detectably. We conclude that (a) A₁AR is unnecessary for LAD2 degranulation or AR enhancement; (b) A_2A, A_2B, and A₃ ARs all contribute to pharmacologic AR enhancement of LAD2 and BMMC degranulation; and (c) LAD2 cells depend on microenvironmental adenosine to trigger AR modulation.     

Endogenous synthesis of prostacyclin was positively regulated during the maturation phase of cultured adipocytes

Prostacyclin alternatively called prostaglandin (PG) I₂ is an unstable metabolite synthesized by the arachidonate cyclooxygenase pathway. Earlier studies have suggested that prostacyclin analogues can act as a potent effector of adipose differentiation. However, biosynthesis of PGI₂ has not been determined comprehensively at different life stages of adipocytes. PGI₂ is rapidly hydrolyzed to the stable product, 6-keto-PGF_1α, in biological fluids. Therefore, the generation of PGI₂ can be quantified as the amount of 6-keto-PGF_1α. In this study, we attempted to develop a solid-phase enzyme-linked immunosorbent assay (ELISA) using a mouse antiserum specific for 6-keto-PGF_1α. According to the typical calibration curve of our ELISA, 6-keto-PGF_1α can be quantified from 0.8 pg to 7.7 ng in an assay. The evaluation of our ELISA revealed the higher specificity of our antiserum without the cross-reaction with other related prostanoids while it exhibited only the cross-reaction of 1.5 % with PGF_2α. The resulting ELISA was applied to the quantification of 6-keto-PGF_1α generated endogenously by cultured 3T3-L1 cells at different stages. The cultured cells showed the highest capability to generate 6-keto-PGF_1α during the maturation phase of 4–6 days, which was consistent with the coordinated changes in the gene expression of PGI synthase and the IP receptor for PGI₂. Following these events, the accumulation of fats was continuously promoted up to 14 days. Thus, our immunological assay specific for 6-keto-PGF_1α is useful for monitoring the endogenous levels of the unstable parent PGI₂ at different life stages of adipogenesis and for further studies on the potential association with the up-regulation of adipogenesis in cultured adipocytes.     

Excretion of primary prostanoids and their metabolites during acute volume expansion

Simultaneous determination of urinary excretion rates of primary unmetabolized prostanoids and their enzymatic metabolites were performed by gas chromatography-mass spectrometry (GC/MS) or tandem mass spectrometry (GC/MS/MS). Changes in kidney function were induced by acute (4 h) volume expansion. Despite marked changes in urine flow, GFR, urinary pH, osmolality, sodium and potassium excretion, only a insignificant or transient rise in the enzymatic prostanoid metabolites (2,3-dinor-6-keto-PGF_1α, PGE-M, 2,3-dinor-TxB₂ and 11-dehydro-TxB₂) was observed. The excretion rates of the primary prostanoids were elevated in parallel with the rise in urine flow: PGE₂ rose (p < 0.05) from 14.2 ± 4.0 to 86.2 ± 20.7, PGF_2α from 60.0 ± 4.9 to 119.8 ± 24.0, 6-keto-PGF_2α from 7.2 ± 1.3 to 51.5 ± 17.0, and txB₂ from 11.2 ± 3.3 to 13.6 ± 3.6 ng/h/1.73 m² ( ) at the maximal urine flow. Except for 6-keto-PGF_1α and TxB₂, this rise in urinary prostanoid levels was only transient despite a sustained fourfold elevated urine flow. We conclude that urine flow rate acutely affect urine prostanoid excretion rates, however, over a prolonged peroid of time these effects are not maintained. The present data support the concept that urinary levels of primary prostanoids mainly reflect renal concentrations whereas those of enzymatic metabolites reflect systemic prostanoid activity. From the excretion pattern of TxB₂ one can assume that this prostanoid represents renal as well as systemic TxA₂ activity.     

COX-derived prostanoid pathways in gastrointestinal cancer development and progression: Novel targets for prevention and intervention

Arachidonic acid metabolism through cyclooxygenase (COX) pathways leads to the generation of biologically active eicosanoids. Eicosanoid expression levels vary during development and progression of gastrointestinal (GI) malignancies.COX-2 is the major COX-isoform responsible for G.I. cancer development/progression. COX-2 expression increases during progression from a normal to cancerous state. Evidence from observational studies has demonstrated that chronic NSAID use reduces the risk of cancer development, while both incidence and risk of death due to G.I. cancers were significantly reduced by daily aspirin intake. A number of randomized controlled trials (APC trial, Prevention of Sporadic Adenomatous Polyps trial, APPROVe trial) have also shown a significant protective effect in patients receiving selective COX-2 inhibitors. However, chronic use of selective COX-2 inhibitors at high doses was associated with increased cardiovascular risk, while NSAIDs have also been associated with increased risk. More recently, downstream effectors of COX-signaling have been investigated in cancer development/progression. PGE₂, which binds to both EP and PPAR receptors, is the major prostanoid implicated in the carcinogenesis of G.I. cancers. The role of TXA₂ in G.I. cancers has also been examined, although further studies are required to uncover its role in carcinogenesis. Other prostanoids investigated include PGD₂ and its metabolite 15d-PGJ2, PGF_1α and PGI₂. Targeting these prostanoids in G.I. cancers has the promise of avoiding cardiovascular toxicity associated with chronic selective COX-2 inhibition, while maintaining anti-tumor reactivity.A progressive sequence from normal to pre-malignant to a malignant state has been identified in G.I. cancers. In this review, we will discuss the role of the COX-derived prostanoids in G.I. cancer development and progression. Targeting these downstream prostanoids for chemoprevention and/or treatment of G.I. cancers will also be discussed. Finally, we will highlight the latest pre-clinical technologies as well as avenues for future investigation in this highly topical research field.     

Altered prostanoid formation in human umbilical vasculature in response to variations in oxygen tension

Prostanoid formation in human umbilical vessels perfused was assessed at different oxygen tensions. At an atmosphere of 5% oxygen the production rate of prostacyclin (measured as 6-keto-PGF_1α) was higher, while those of thromboxane A₂ (measured as TXB₂), PGE₂ and PGF_2α were lower than with 20%, 50% and 95% oxygen. The stimulatory effect of angiotensin II on prostanoid production was found to be independent on the prevailing oxygen tension. Vascular formation of prostanoids thus seems to be at least partially affected by the ambient oxygen tension. Though altered oxygen tension does not seen to affect angiotensin induced prostanoid formation, the action of other vasoactive agents influencing vascular formation of prostanoids may respond differently to hypoxia or hyperoxia.     

Characterization of prostaglandin F2α receptor of mouse 3T3 fibroblasts and its functional expression in Xenopus laevis oocytes

Prostaglandin (PG) F_2α increased [³H]thymidine incorporation into quiescent NIH 3T3 cells, stimulated phosphoinositide breakdown, and raised intracellular Ca²⁺ concentration ([Ca²⁺]_i) in a dose-dependent manner with ED₅₀ values of 2.0 × 10^?8 M, 4.6 × 10^?8 M, and 7.5 × 10^?8 M, respectively. The increase in [³H]thymidine incorporation with PGF_2α was additive with that seen with epidermal growth factor (EGF) or insulin. The peak [Ca²⁺]_i increase with PGF_2α was still obvious in the absence of extracellular Ca²⁺ and was insensitive to islet activating protein (IAP) pretreatment. Membranes prepared from NIH 3T3 cells exhibited a specific binding for PGF_2α, which was sensitive to GTP_γS but not sensitive to IAP pretreatment. Xenopus laevis oocytes injected with NIH 3T3 cell mRNA between 18S and 28S rRNA fractionated by sucrose gradient, expressed a PGF_2α-specific Cl^? current when examined by voltage clamp. This Cl^? current was also insensitive to IAP pretreatment and not affected by extracellular Ca²⁺ concentration ([Ca²⁺]_o). These results indicate 1) that the NIH 3T3 cells expressed a specific PGF_2α receptor which is linked to phosphoinositide-specific phospholipase C (PLC) activation and to mobilization of Ca²⁺ via an IAP-insensitive G-proteins(s), 2) that this PGF_2α receptor may play an active role in the proliferation of NIH 3T3 cells, and 3) that this PGF_2α receptor can be expressed in the oocyte system. © 1993 Wiley-Liss, Inc.