Prostanoid EP1 receptors mediate up-regulation of the orphan nuclear receptor Nurr1 by cAMP-independent activation of protein kinase A, CREB and NF-κB.

BACKGROUND AND PURPOSE: Prostaglandin E(2) (PGE(2)) stimulation of the G protein-coupled prostanoid EP(1) receptor was found to up-regulate the expression of Nur-related factor 1 (Nurr1) (NR4A2), a transcription factor in the NR4A subfamily of nuclear receptors. The present studies characterize the molecular mechanism of this up-regulation. EXPERIMENTAL APPROACH: The expression of Nurr1 was examined by immunoblot analysis, the polymerase chain reaction and reporter gene assays in human embryonic kidney (HEK) cells stably expressing the recombinant EP(1) receptor and in SH-SY5Y neuroblastoma cells expressing endogenous EP(1) receptors. Signalling pathway inhibitors were used to examine the roles of Rho, PKA, the cAMP response element binding protein (CREB) and NF-κB on the PGE(2) stimulated up-regulation of Nurr1. CREB and NF-κB signalling were also examined by immunoblot analysis and reporter gene assays. KEY RESULTS: The EP(1) receptor mediated up-regulation of Nurr1 was blocked with inhibitors of Rho, PKA, NF-κB and CREB; but PGE(2) failed to significantly stimulate intracellular cAMP formation. PGE(2) stimulation of the EP1 receptor induced the phosphorylation and activation of CREB and NF-κB, which could be blocked by inhibition of PKA. CONCLUSIONS AND IMPLICATIONS: PGE(2) stimulation of the human EP(1) receptor up-regulates the expression of Nurr1 by a mechanism involving the sequential activation of the Rho, PKA, CREB and NF-κB signalling pathways. EP(1) receptors are implicated in tumorigenesis and the up-regulation of Nurr1 may underlie the anti-apoptotic effects of PGE(2) .

Activation of the human FP prostanoid receptor disrupts mitosis progression and generates aneuploidy and polyploidy.

Studies have shown prostaglandin F(2alpha) (PGF(2alpha)) to be an endogenous tumor promoter in mouse models of skin carcinogenesis; however, the mechanisms by which PGF(2alpha) affects cell cycle events remain unknown. Here we performed cell cycle analyses on HEK cells stably expressing the human FP receptor and found that treatment with PGF(2alpha) delays mitosis and is associated with an increased expression of cyclin B1 and Cdc2 kinase activity. In addition, multipolar spindles and misaligned chromosomes were observed in a significant proportion of cells treated with PGF(2alpha). Defective cytokinesis was also observed which resulted in gross aneuploidy and polyploidy. Expression of dominant negative Rho attenuated the cell cycle delay and prevented the generation of micronuclei following treatment with PGF(2alpha). This suggests that FP receptor activation of Rho signaling by PGF(2alpha) can interfere with nuclear division. Aneuploidy is associated with genomic instability and may underlie the tumor-promoting properties of PGF(2alpha).

The growth of malignant keratinocytes depends on signaling through the PGE(2) receptor EP1.

Recent discoveries shed light on the importance of prostaglandin (PG) production in the development of skin cancer. Work by Fischer et al. demonstrates that skin tumor promotion caused by ultraviolet B radiation can be decreased by up to 89% by blocking cyclooxygenase-2 (COX-2) with the drug Celecoxib. A similar study showed that Celecoxib can decrease new tumor formation by 44% in mice that already have tumors. These studies demonstrate the importance of COX-2 and PGs in the development of squamous cell carcinoma. We have explored growth signaling in a model of skin tumor progression. Because changes in PG production have been implicated in skin carcinogenesis, we examined this pathway. We found that malignant cell lines secrete more prostaglandin E(2) (PGE(2)) than the parental cells. We observed increased expression of COX-1 and -2. We also found that these cells express the PGE(2) receptors EP1 and EP4. When the cells are grown in the presence of indomethacin, the growth rate of the malignant cells is decreased. This effect can be reversed by addition of PGE(2) or an EP1 agonist to the medium. Thus, we have shown that skin tumor cells depend in part on PGE(2) signaling through the EP1 prostanoid receptor for their in vitro growth.

Receptor number and caveolar co-localization determine receptor coupling efficiency to adenylyl cyclase.

Recent evidence suggests that many signaling molecules localize in microdomains of the plasma membrane, particularly caveolae. In this study, overexpression of adenylyl cyclase was used as a functional probe of G protein-coupled receptor (GPCR) compartmentation. We found that three endogenous receptors in neonatal rat cardiomyocytes couple with different levels of efficiency to the activation of adenylyl cyclase type 6 (AC6), which localizes to caveolin-rich membrane fractions. Overexpression of AC6 enhanced the maximal cAMP response to beta(1)-adrenergic receptor (beta(1)AR)-selective activation 3.7-fold, to beta(2)AR-selective activation only 1.6-fold and to prostaglandin E(2) (PGE(2)) not at all. Therefore, the rank order of efficacy in coupling to AC6 is beta(1)AR > beta(2)AR > prostaglandin E(2) receptor (EP(2)R). beta(2)AR coupling efficiency was greater when we overexpressed the receptor or blocked its desensitization by expressing betaARKct, an inhibitor of G protein-coupled receptor kinase activation, but was not significantly greater when cells were treated with pertussis toxin. Assessment of receptor and AC expression indicated co-localization of AC5/6, beta(1)AR, and beta(2)AR, but not EP(2)R, in caveolin-rich membranes and caveolin-3 immunoprecipitates, likely explaining the observed activation of AC6 by betaAR subtypes but lack thereof by PGE(2). When cardiomyocytes were stimulated with a betaAR agonist, beta(2)AR were no longer found in caveolin-3 immunoprecipitates; an effect that was blocked by expression of betaARKct. Thus, agonist-induced translocation of beta(2)AR out of caveolae causes a sequestration of receptor from effector and likely contributes to the lower efficacy of beta(2)AR coupling to AC6 as compared with beta(1)AR, which do not similarly translocate. Therefore, spatial co-localization is a key determinant of efficiency of coupling by particular extracellular signals to activation of GPCR-linked effectors.

FP prostanoid receptor activation of a T-cell factor/beta -catenin signaling pathway.

FP prostanoid receptors are G-protein-coupled receptors (GPCR) that consist of two known isoforms, FP(A) and FP(B). These isoforms, which are generated by alternative mRNA splicing, are identical except for their carboxyl-terminal domains. Previously we have shown that stimulation of both isoforms with prostaglandin F(2alpha) (PGF(2alpha)) activates the small G-protein Rho, leading to morphological changes consisting of cell rounding and the formation of cell aggregates. Following the removal of PGF(2alpha), however, FP(A)-expressing cells show rapid reversal of cell rounding, whereas FP(B)-expressing cells do not. We now show that acute treatment of FP(B)-expressing cells with PGF(2alpha) leads to a subcellular reorganization of beta-catenin, a decrease in the phosphorylation of cytoplasmic beta-catenin, and persistent stimulation of Tcf/Lef-mediated transcriptional activation. This does not occur in FP(A)-expressing cells and may underlie the differences between these isoforms with respect to the reversal of cell rounding. The Tcf/beta-catenin signaling pathway is known to mediate the actions of Wnt acting through the heptahelical receptor, Frizzled, and has not been associated previously with GPCR activation. Our findings expand the signaling possibilities for GPCRs and suggest novel roles for FP receptors in normal tissue development and malignant transformation.

Replacement of the carboxylic acid group of prostaglandin f(2alpha) with a hydroxyl or methoxy substituent provides biologically unique compounds.

Replacement of the carboxylic acid group of PGF(2alpha) with the non-acidic substituents hydroxyl (-OH) or methoxy (-OCH(3)) resulted in an unexpected activity profile. Although PGF(2alpha) 1-OH and PGF(2alpha) 1-OCH(3) exhibited potent contractile effects similar to 17-phenyl PGF(2alpha) in the cat lung parenchymal preparation, they were approximately 1000 times less potent than 17-phenyl PGF(2alpha) in stimulating recombinant feline and human FP receptors. In human dermal fibroblasts and Swiss 3T3 cells PGF(2alpha) 1-OH and PGF(2alpha) 1-OCH(3) produced no Ca(2+) signal until a 1 microM concentration was exceeded. Pretreatment of Swiss 3T3 cells with either 1 microM PGF(2alpha) 1-OH or PGF(2alpha) 1-OCH(3) did not attenuate Ca(2+) signal responses produced by PGF(2alpha) or fluprostenol. In the rat uterus, PGF(2alpha) 1-OH was about two orders of magnitude less potent than 17-phenyl PGF(2alpha) whereas PGF(2alpha) 1-OCH(3) produced only a minimal effect. Radioligand binding studies on cat lung parenchymal plasma membrane preparations suggested that the cat lung parenchyma does not contain a homogeneous population of receptors that equally respond to PGF(2alpha)1-OH, PGF(2alpha)1-OCH(3), and classical FP receptor agonists. Studies on smooth muscle preparations and cells containing DP, EP(1), EP(2), EP(3), EP(4), IP, and TP receptors indicated that the activity of PGF(2alpha) 1-OH and PGF(2alpha) 1-OCH(3) could not be ascribed to interaction with these receptors. The potent effects of PGF(2alpha) 1-OH and PGF(2alpha) 1-OCH(3) on the cat lung parenchyma are difficult to describe in terms of interaction with the FP or any other known prostanoid receptor.

Delayed reversal of shape change in cells expressing FP(B) prostanoid receptors. Possible role of receptor resensitization.

Prostaglandin F(2 alpha) (PGF(2 alpha)) receptors are G-protein-coupled receptors consisting of two alternative mRNA splice variants, named FP(A) and FP(B). As compared with the FP(A) isoform, the FP(B) isoform lacks the last 46 amino acids of the carboxyl terminus and, therefore, represents a truncated version of the FP(A). We recently found (Pierce, K. L., Fujino, H., Srinivasan, D., and Regan, J. W. (1999) J. Biol. Chem. 274, 35944-35949) that stimulation of both isoforms with PGF(2 alpha) leads to activation of a Rho signaling pathway, resulting in tyrosine phosphorylation of p125 focal adhesion kinase, formation of actin stress fibers, and cell rounding. Although the activation of Rho and subsequent cell rounding occur at a similar rate for both isoforms, we now report that following the removal of PGF(2 alpha) the reversal of cell rounding is much slower for cells expressing the FP(B) isoform as compared with the FP(A) isoform. Thus, in HEK-293 cells that stably express the FP(A) isoform, the reversal of cell rounding appears to be complete after 1 h, whereas for FP(B)-expressing cells there is essentially no reversal even after 2 h. Similarly, the disappearance of stress fibers and dephosphorylation of p125 focal adhesion kinase following removal of agonist are much slower in FP(B)-expressing cells than in FP(A)-expressing cells. The mechanism of this differential reversal appears to involve a difference in receptor resensitization following the removal of agonist. Based upon whole cell radioligand binding, agonist-induced stimulation of inositol phosphate formation, and mobilization of intracellular Ca(2+), the FP(B) isoform resensitizes more slowly than the FP(A) isoform. These findings suggest that the carboxyl terminus of the FP(A) is critical for resensitization and that the slower resensitization of the FP(B) isoform leads to prolonged signaling. This differential signaling distinguishes the FP(A) and FP(B) receptor isoforms and could be important toward understanding the physiological actions of PGF(2 alpha).

Characterization of ep prostanoid receptor subtypes in primary cultures of bovine ciliary epithelial cells by immunofluorescent microscopy and functional studies.

PURPOSE: To determine the expression and functional coupling of EP prostanoid receptor subtypes to second messenger pathways in bovine ciliary epithelium. METHODS: Primary cultures of bovine ciliary epithelial (BCE) cells were established and maintained in culture up to four passages. EP receptor protein expression was examined by indirect immunofluorescence microscopy with subtype selective antibodies in both tissue sections and primary cultures of BCE cells. Messenger RNA expression was determined using reverse transcription and polymerase chain reaction. The effects of prostanoid agonists on total inositol phosphate accumulation and cAMP formation were used to assess functional activity. RESULTS: Positive immunoreactivity was obtained in both frozen thin-sections and primary cultures of bovine ciliary process to the prostanoid EP(1 ), EP(2), EP(3) and EP(4) receptor subtypes. Reverse transcription followed by the polymerase chain reaction yielded products corresponding to each of the prostanoid EP subtypes which was confirmed by restriction enzyme analysis. PGE(2) dose-dependently stimulated the accumulation of total inositol phosphates in cultured cells with an EC( 50) value of 100 nM. PGE(2), forskolin and isoproterenol produced dose-dependent increases in cAMP formation with EC(50) values of 100, 300 and 200 nM, respectively. Isoproterenol-stimulated cAMP formation was attenuated by the EP(3) receptor agonist sulprostone in cultured BCE cells. The inhibition elicited by sulprostone was reversed in cells pretreated with pertussis toxin. CONCLUSIONS: This study demonstrates the presence of functional prostanoid EP receptor subtypes in the bovine ciliary epithelium. EP(1) and EP(4) receptor subtypes were found primarily in the NPE cells, whereas, EP(2) receptor subtype immunofluorescence was detected in the PE cells. EP(3) receptor subtype labeling was observed in both the NPE and the PE cells. PGE(2) produces opposing effects on adenylyl cyclase through EP(2)/EP(4) and EP(3) receptor activation. The predominant effect of PGE(2) is on the adenylyl cyclase stimulatory receptors (EP(2)/EP(4)).

Inhibition of aquaporin-1 water permeability by tetraethylammonium: involvement of the loop E pore region.

Previously, the only known blockers of water permeability through aquaporin-1 (AQP1) water channels were mercurial reagents such as HgCl(2). For AQP1, inhibition by mercury has been attributed to the formation of a mercaptide bond with cysteine residue 189 found in the putative pore-forming region loop E. Here we show that the nonmercurial compound, tetraethylammonium (TEA) chloride, reduces the water permeability of human AQP1 channels expressed in Xenopus oocytes. After preincubation of the oocytes for 15 min with 100 microM TEA, AQP1 water permeability was reduced by 20 to 40%, a degree of partial block similar to that obtained with 15 min of incubation in 100 microM HgCl(2). The reduction of water permeability was dose-dependent for tested concentrations up to 10 mM TEA. TEA blocks the Shaker potassium channel by interacting with a tyrosine residue in the outer pore region. We tested whether an analogous tyrosine residue in loop E of AQP1 could be involved in the binding of TEA. Using polymerase chain reaction, tyrosine 186 in AQP1, selected for its proximity to the mercury-binding site, was mutated to phenylalanine (Y186F), alanine (Y186A), or asparagine (Y186N). Oocyte expression of the mutant AQP1 channels showed that the water permeability of Y186F was equivalent to that of wild-type AQP1; the other mutant channels did not conduct water. However, in contrast to wild-type AQP1, the water permeability of Y186F was not reduced with 100 microM TEA. These results suggest that TEA reduces AQP1 water permeability by interacting with loop E.

Cloned human aquaporin-1 is a cyclic GMP-gated ion channel.

Aquaporin-1 (AQP1) is a member of the membrane intrinsic protein (MIP) gene family and is known to provide pathways for water flux across cell membranes. We show here that cloned human AQP1 not only mediates water flux but also serves as a cGMP-gated ion channel. Two-electrode voltage-clamp analyses showed consistent activation of an ionic conductance in wild-type AQP1-expressing oocytes after the direct injection of cGMP (50 nl of 100 mM). Current activation was not observed in control (water-injected) oocytes or in AQP5-expressing oocytes with osmotic water permeabilities equivalent to those seen with AQP1. Patch-clamp recordings revealed large conductance channels (150 pS in K(+) saline) in excised patches from AQP1-expressing oocytes after the application of cGMP to the internal side. Amino acid sequence alignments between AQP1 and sensory cyclic-nucleotide-gated channels showed similarities between the cyclic-nucleotide-gated binding domain and the AQP1 carboxyl terminus that were not present in AQP5. Competitive radioligand-binding assays with [(3)H]cGMP demonstrated specific binding (K(D) = 0.2 microM) in AQP1-expressing Sf9 cells but not in controls. These results indicate that AQP1 channels have the capacity to participate in ionic signaling after the activation of cGMP second-messenger pathways.

Differential regulation of prostaglandin F(2alpha) receptor isoforms by protein kinase C.

Prostaglandin F(2alpha) receptors (FP) are G protein-coupled receptors that bind prostaglandin F(2alpha) (PGF(2alpha)), resulting in the activation of an inositol phosphate (IP) second messenger pathway. Alternative mRNA splicing generates two FP receptor isoforms. These isoforms, designated FP(A) and FP(B), are otherwise identical except for their carboxyl termini. FP(B) is essentially a truncated version of FP(A) that lacks the 46 carboxyl-terminal amino acids, including four putative protein kinase C (PKC) phosphorylation sites. Until now, functional differences between these FP receptor isoforms have not been identified. We now report that pretreatment with the PKC inhibitor bisindolylmaleimide I enhanced PGF(2alpha)-stimulated IP accumulation in transfected cells stably expressing the FP(A) isoform but not in cells stably expressing the FP(B) isoform. Whole-cell phosphorylation experiments showed a strong agonist-dependent phosphorylation of the FP(A) isoform but little or no phosphorylation of the FP(B). Pretreatment of cells with bisindolylmaleimide I decreased PGF(2alpha)-stimulated phosphorylation of the FP(A) isoform consistent with a PKC-dependent phosphorylation. In vitro phosphorylation of an FP(A) carboxyl-terminal fusion protein by recombinant PKCalpha showed that the carboxyl terminus of the FP(A) is a substrate for PKC. These results suggest that PKC-dependent phosphorylation is responsible for differential regulation of second messenger signaling by FP prostanoid receptor isoforms.

Activation of FP prostanoid receptor isoforms leads to Rho-mediated changes in cell morphology and in the cell cytoskeleton.

Prostaglandin F(2alpha) (PGF(2alpha)) exerts its biological effects by binding to and activating FP prostanoid receptors. These receptors, which include two isoforms, the FP(A) and FP(B), have been cloned from a number of species and are members of the superfamily of G-protein-coupled receptors. Previous studies have shown that the activation of FP receptors leads to phosphatidylinositol hydrolysis, intracellular calcium release, and activation of protein kinase C. Here, we demonstrate that PGF(2alpha) treatment of 293-EBNA (Epstein-Barr nuclear antigen) cells that have been stably transfected with either the FP(A) or FP(B) receptor isoforms leads to changes in cell morphology and in the cell cytoskeleton. Specifically, cells treated with PGF(2alpha) show retraction of filopodia and become rounded, and actin stress fibers are formed. Pretreatment of the cells with bisindolylmaleimide I, a protein kinase C inhibitor, has no effect on the PGF(2alpha)-induced changes in cell morphology, although it does block the effects of phorbol myristate acetate on cell morphology. On the other hand, the PGF(2alpha)-induced changes in cell morphology and formation of actin stress fibers can be blocked by pretreatment of the cells with C3 exoenzyme, a specific inhibitor of the small G-protein, Rho. Consistent with FP receptor induced formation of actin stress fibers and focal adhesions, FP(A) receptor activation also leads to rapid (within two minutes) tyrosine phosphorylation of p125 focal adhesion kinase (FAK) which can be blocked by pretreating the cells with C3 exoenzyme. Taken together, these results suggest that the FP receptor isoforms are coupled to at least two second messenger pathways, one pathway associated with protein kinase C activation, and the other with activation of Rho.

Changes in the expression of alpha(2)-adrenergic receptor subtypes during maturation of neuronal cells from fetal pig superior cervical ganglia.

The expression of presynaptic alpha(2)-adrenergic receptor (alpha(2)-AR) subtypes was investigated in cultured neurons from fetal pig superior cervical ganglion (SCG). Cells were incubated with chicken antibodies against alpha(2)A-, alpha(2)B- or alpha(2)C-AR subtypes either alone or together with antibodies against dopamine-beta-hydroxylase (DbetaH, a marker for adrenergic neurons) or against choline acetyl transferase (ChAT, a marker for cholinergic neurons). We found immunoreactivity for all three alpha(2)-AR subtypes in SCG-cells when cultured for 8-11 days. The relative expression of the alpha(2)A-subtype was approximately 1/3 of that of alpha(2)B- and alpha(2)C-AR. Co-localisation of all three alpha(2)-AR subtypes was observed in cells expressing DbetaH or ChAT. Increasing the potassium concentration in the culture medium increased the expression of DbetaH and decreased the expression of the alpha(2)A- and alpha(2)C-subtype without altering the expression of the alpha(2)B-subtype. Co-culture of neurons with pig splenocytes enhanced the expression of ChAT and decreased the expression of the alpha(2)B-subtype without altering the expression of alpha(2)A- and alpha(2)C-subtypes. Our results indicate that the three alpha(2)-receptor subtypes are expressed on both noradrenergic and cholinergic nerves. Induction of the noradrenergic phenotype favours the expression of the alpha(2)B-subtype over that of the alpha(2)A- and alpha(2)C-subtype. Conversely, enhancement of the cholinergic phenotype favours the expression of the alpha(2)A- and alpha(2)C-subtypes over that of the alpha(2)B-subtype. Our results suggest that the alpha(2)B-receptor is preferentially associated with noradrenergic nerve endings.

Ovine prostaglandin F2alpha receptor: steroid influence on steady-state levels of luteal mRNA.

Expression of the receptor for prostaglandin F2alpha (PGF2alpha) is decreased in the ovine corpus luteum during regression and increased in early pregnancy. This study was designed to evaluate the influence of progesterone and/or 17beta-estradiol (E2) on this regulation. Circulating progesterone (functional regression) and luteal PGF receptor mRNA decreased (p < 0.05) within 8 h of PGF2alpha-induced luteal regression in midluteal phase (day 10; d 10) ewes; however, internucleosomal DNA fragmentation (structural regression) was not yet increased. Additionally, luteal PGF receptor mRNA and circulating progesterone were greater (p < 0.05) in pregnant than in nonpregnant ewes on d 14, but not on d 12. Twelve hours following injection of d 10 ewes with E2, steady-state levels of mRNA for PGF receptor were decreased (p < 0.05), although circulating progesterone and DNA laddering were unchanged. Conversely, luteal mRNA for PGF receptor was increased (p < 0.05) by E2 treatment in hysterectomized ewes. These results provide evidence that (1) luteal PGF receptor expression parallels circulating progesterone levels during functional regression and in early pregnancy, but (2) expression of PGF receptor can be dissociated from alterations in circulating progesterone by injection with E2. Additionally, decreased PGF receptor expression initiated by E2 is uterine-dependent, whereas the direct luteal effect (hysterectomized ewes) of E2 is a stimulation of PGF receptor expression. These results collectively support the belief that the apparent downregulation of PGF receptor during luteal regression is associated with uterine-derived PGF2alpha and its intracellular effects rather than with alterations in ovarian steroid production.

A single amino-acid substitution in the EP2 prostaglandin receptor confers responsiveness to prostacyclin analogs.

A high degree of homology between the four Gs-coupled prostaglandin (PG) receptors [EP2, EP4, prostacyclin (IP), PGD2 (DP)] and the four Gq/Gi-coupled receptors [EP1, EP3, PGF2alpha (FP), thromboxane A2 (TP)] suggests that prostaglandin receptors evolved functionally from an ancestral EP receptor before the development of distinct binding epitopes. If so, ligand selectivity should be determined by a limited number of amino acids. EP2 receptor transmembrane domain residues that are similar to those in the EP4 receptor but differ from those in the IP receptor were mutated to the corresponding IP receptor residue. Activation of the mutant receptors by PGE2 (EP2 ligand), iloprost (stable prostacyclin analog), and PGE1 (EP2/IP ligand) was determined using a cAMP-dependent reporter gene assay. A Leu304-to-tyrosine substitution in the seventh transmembrane domain enhanced iloprost potency approximately 100-fold. A glycine substitution at Ser120 in the third transmembrane domain had no effect on drug potency but improved the response of the Tyr304 mutant. The potency of the natural prostaglandins PGF2alpha and PGD2 was not enhanced by the mutations. In contrast, the potency of all prostaglandins was reduced 10- to 100-fold when arginine 302, which is thought to be a counterion for the prostaglandin carboxylic acid, was mutated. Thus, a single amino acid change resulted in a selective gain of function for iloprost, which is consistent with the proposed phylogeny of the prostaglandin receptors.

Prostanoid receptor heterogeneity through alternative mRNA splicing.

Prostaglandin (PG) and thromboxane (TX) receptors are G-protein coupled receptors that mediate the physiological actions of the five principal prostanoid metabolites: PGD2, PGE2, PGF2alpha, PGI2 (prostacyclin) and TXA2. Five major subdivisions of the prostanoid receptor family have been defined pharmacologically which correspond to each of the metabolites as follows: DP, EP, FP IP and TP. The EP receptors have been further classified pharmacologically into the EP1, EP2, EP3 and EP4 subtypes. Molecular biological studies have resulted in the cloning of cDNA's encoding all of these prostanoid receptors. In addition, the cloning of these receptors has revealed further heterogeneity through the use of alternative mRNA splicing. Specifically, mRNA splice variants have been identified for the EP1, EP3, FP and TP receptors. Interestingly, except for the EP1 receptors, the mechanisms giving rise to these receptor isoforms involves the use of splice sites located in the cytoplasmic carboxyl termini of these receptors. Thus, the eight human EP3 isoforms that have been identified are otherwise identical except for their carboxyl termini. Similarly, the optional use of a potential splice site encoding the carboxyl terminus gives rise to each of the two FP and TP receptor isoforms. Because the carboxyl termini of G-protein coupled receptors are generally implicated in interactions with G-proteins, it is not surprising that these receptor isoforms differ mainly with respect to their activation of second messenger pathways and not in their pharmacological characteristics. Differences also exist with respect to their levels of constitutive activity (e.g., in the absence of agonist) and in their desensitization.

Alpha 2-adrenergic receptors increase cell migration and decrease F-actin labeling in rat aortic smooth muscle cells.

Vascular wound healing and such pathologies as atherosclerosis and restenosis are characterized by migration and proliferation of the smooth muscle cells of the media after denudation of the intima. To explore possible roles that alpha 2-adrenergic receptors (alpha 2-ARs) might have in these cellular responses, we characterized the alpha 2-ARs present in explant-derived cultures of rat aortic smooth muscle (RASM) cells. The results of immunofluorescence microscopy and reverse transcription followed by the polymerase chain reaction indicated that all three alpha 2-AR subtypes (alpha 2A, alpha 2B, and alpha 2C) were initially present. Mitogen-activated protein kinase activity in the RASM cells was stimulated fivefold over basal by the alpha 2-selective agonist dexmedetomidine (Dex) and was blocked by coincubation with the alpha 2-selective antagonist rauwolscine (RW) or by preincubation of the cells with the Gi/G(o)-protein inhibitor pertussis toxin. alpha 2-AR activation by Dex did not promote cell proliferation, as measured by the incorporation of [3H]thymidine. However, Dex significantly increased RASM cell migration, and antagonist blocked this effect. Incubation of RASM cells with Dex also produced a marked decrease in F-actin labeling, which again was prevented by coincubation with RW. The evidence clearly reveals the presence of functional alpha 2-ARs in RASM cells. The involvement of alpha 2-AR activation with cytoskeletal changes and cell migration is novel and indicates a potential role of these receptors in vascular wound healing and pathogenesis.

Prostaglandin F2 alpha receptors in the human trabecular meshwork.

PURPOSE: Prostaglandin F2 alpha (PGF2 alpha) and analogs, such as latanoprost, are thought to lower intraocular pressure (IOP), primarily by increasing uveoscleral outflow. However, outflow through the trabecular meshwork may be increased as well. The authors hypothesize that any effect on the trabecular meshwork is mediated by prostanoid FP receptors (receptors for prostaglandin F2 alpha) in this tissue. METHODS: To test this hypothesis, tissue sections of the human trabecular meshwork and cultures of human trabecular meshwork cells were examined for the presence of FP receptors using immunofluorescence microscopy with affinity-purified antibodies raised against a glutathione-S-transferase (GST)-FPA receptor fusion protein. The presence of the receptor was confirmed by using reverse transcription-polymerase chain reaction (RT-PCR), functional assays of PGF2 alpha-stimulated inositol phosphate hydrolysis, and intracellular calcium measurements. RESULTS: Positive FPA receptor immunolabeling was observed in sections of the human trabecular meshwork and in cultured human trabecular meshwork cells. In both cases, specific labeling could be blocked by preincubation with a GST-FPA receptor fusion protein. Cross-blocking experiments with other receptor fusion proteins did not block specific labeling in cultured trabecular meshwork cells. PGF2 alpha caused a dose-dependent increase in total inositol phosphate accumulation and intracellular calcium release in human trabecular meshwork cells that was consistent with the presence of FP receptors. Using RT-PCR, message-encoding prostanoid FPA receptors were found in total RNA isolated from human trabecular meshwork cells. CONCLUSIONS: Prostanoid FPA receptors exist in human trabecular meshwork cells, as shown by the presence of mRNA, protein, and functional response to PGF2 alpha. This study indicates that functional FP receptors are present in the human trabecular meshwork and that they may be involved in mediating some of the IOP-lowering effects of PGF2 alpha in the eye.

Evidence for platelet-activating factor receptor subtypes on human polymorphonuclear leukocyte membranes.

Platelet-activating factor (PAF) is a potent phospholipid mediator that acts through specific cell surface receptors. The existence of PAF receptor subtypes has been suggested by functional and radioligand binding studies in a variety of cells and tissues. This report addresses this issue more directly and demonstrates differences between specific PAF receptors in human polymorphonuclear leukocytes (PMNs) and COS-7 cells transfected with the cloned human PAF receptor gene. The presence of more than one receptor in human PMNs is supported by three different studies. First, the Kd from the saturation isotherms for the binding of [3H]WEB 2086 on PMNs was 7-fold larger (Kd = 29.2 nM) than the kinetic Kd (4.2 nM). Second, the pseudo-Hill slope determined from the saturation experiments with PMNs was significantly lower than unity (0.69 +/- 0.05 SEM), and the saturation Kd values for transfected COS-7 (Kd = 9.6 nM) and PMN membranes were significantly different. These results contrasted with those for the transfected COS-7 cells, which showed a Kd from the saturation isotherms similar to that of the kinetic Kd (3.2 nM) and a pseudo-Hill slope that was not different from 1.0. Third, when the radiolabeled ligand [3H]WEB 2086 was increased in concentration from 10 to 50 nM in inhibition experiments with the human PMN membranes, the Ki increased, indicative of binding mainly to receptors with lower affinity. These results suggest that PAF receptor subtypes exist in human PMNs based on distinct radioligand binding characteristics from the human cloned PAF receptor.