Complement C5a stimulates food intake via a prostaglandin D(2)- and neuropeptide Y-dependent mechanism in mice.

We have recently found that prostaglandin (PG) D(2) stimulates food intake via DP(1) receptor. Here we show that complement C5a stimulates food intake by activating the orexigenic PGD(2) system. C5a (30-100 pmol/mouse), after intracerebroventricular administration, stimulated food intake in non-food-deprived mice. The orexigenic activity of C5a was blocked by co-administration of a DP(1) receptor antagonist, BWA868C. Central administration of C5a elevated the hypothalamic mRNA expression of COX-2 but not COX-1, and the food intake stimulation of C5a was inhibited by pretreatment with a COX-2 inhibitor, celecoxib, suggesting that C5a activates COX-2 upstream of the PGD(2)-DP(1) system. The orexigenic activity of C5a was also inhibited by an antagonist for neuropeptide Y (NPY) Y(1) receptor, which was activated downstream of the PGD(2)-DP(1) system. These results suggest that C5a stimulates food intake via a PGD(2)- and NPY-dependent mechanism. C5a is the first example of orexigenic peptides acting through the PGD(2)-NPY system in the central nervous system.

CRTH2 plays an essential role in the pathophysiology of Cry j 1-induced pollinosis in mice.

PGD(2) is the major prostanoid produced during the acute phase of allergic reactions. Two PGD(2) receptors have been isolated, DP and CRTH2 (chemoattractant receptor-homologous molecule expressed on Th2 cells), but whether they participate in the pathophysiology of allergic diseases remains unclear. We investigated the role of CRTH2 in the initiation of allergic rhinitis in mice. First, we developed a novel murine model of pollinosis, a type of seasonal allergic rhinitis. Additionally, pathophysiological differences in the pollinosis were compared between wild-type and CRTH2 gene-deficient mice. An effect of treatment with ramatroban, a CRTH2/T-prostanoid receptor dual antagonist, was also determined. Repeated intranasal sensitization with Cry j 1, the major allergen of Cryptomeria japonica pollen, in the absence of adjuvants significantly exacerbated nasal hyperresponsive symptoms, Cry j 1-specific IgE and IgG1 production, nasal eosinophilia, and Cry j 1-induced in vitro production of IL-4 and IL-5 by submandibular lymph node cells. Additionally, CRTH2 mRNA in nasal mucosa was significantly elevated in Cry j 1-sensitized mice. Following repeated intranasal sensitization with Cry j 1, CRTH2 gene-deficient mice had significantly weaker Cry j 1-specific IgE/IgG1 production, nasal eosinophilia, and IL-4 production by submandibular lymph node cells than did wild-type mice. Similar results were found in mice treated with ramatroban. These results suggest that the PGD(2)-CRTH2 interaction is elevated following sensitization and plays a proinflammatory role in the pathophysiology of allergic rhinitis, especially pollinosis in mice.

Effect of the statin atorvastatin on intracellular signalling by the prostacyclin receptor in vitro and in vivo.

Prostacyclin plays a central role within the vasculature. We have previously established that the prostacyclin receptor (IP) undergoes isoprenylation, a lipid modification obligate for its function. The aim of the current study was to investigate the effect of the hydroxy methyl glutaryl co-enzyme A reductase inhibitor atorvastatin on signalling and function of the IP expressed in mammalian whole cells and in platelets isolated from patients undergoing therapeutic intervention with atorvastatin. Initially, the effect of atorvastatin on signalling by the human (h) and mouse (m) IP overexpressed in human embryonic kidney 293 cells and the hIP endogenously expressed in human erythroleukaemic 92.1.7 cells was investigated. Atorvastatin significantly reduced IP-mediated cAMP generation (IC(50) 6.6-11.1 microm) and [Ca(2+)](i) mobilization (IC(50) 7.2-16.4 microm) in a concentration-dependent manner, but had no effect on signalling by the nonisoprenylated beta(2) adrenergic receptor or the alpha or beta isoforms of the human thromboxane A(2) receptor (TP). Moreover, atorvastatin significantly reduced IP-mediated crossdesensitization of signalling by TP alpha (IC(50) 10.4 microm), but not by TP beta. In contrast to the whole-cell data, atorvastatin therapy did not interfere with IP-mediated cAMP generation or IP-induced inhibition of TP-mediated aggregation of platelets isolated from human volunteers undergoing therapeutic intervention with atorvastatin (10-80 mg per daily dose). In conclusion, while data generated in whole cells indicated that atorvastatin significantly impairs signalling by both the hIP and mP, the in vivo clinical data indicated that, at the administered therapeutic dose, atorvastatin does not significantly compromise IP signalling and function in humans.

Ocular hypotensive FP prostaglandin (PG) analogs: PG receptor subtype binding affinities and selectivities, and agonist potencies at FP and other PG receptors in cultured cells.

Natural prostaglandins (PGs) such as PGD2, PGE2, PGF2(2alpha), and PGI2 exhibited the highest affinity for their respective cognate receptors, but were the least selective agents when tested in receptor binding assays. Travoprost acid ([+]-fluprostenol) was the most FP-receptor-selective compound, exhibiting a high affinity (Ki = 35 +/- 5 nM) for the FP receptor, and minimal affinity for DP (Ki = 52,000 nM), EP1 (Ki = 9540 nM), EP3 (Ki = 3501 nM), EP4 (Ki = 41,000 nM), IP (Ki > 90,000 nM), and TP (Ki = 121,000 nM) receptors. Travoprost acid was the most potent PG analog tested in FP receptor functional phosphoinositide turnover assays in the following cell types: human ciliary muscle (EC50 = 1.4 nM), human trabecular meshwork (EC50 = 3.6 nM), and mouse fibroblasts and rat aortic smooth muscle cells (EC50 = 2.6 nM). Although latanoprost acid exhibited a relatively high affinity for the FP receptor (Ki = 98 nM), it had significant functional activity at FP (EC50 = 32-124 nM) and EP1 (EC50 = 119 nM) receptors. Bimatoprost acid was less selective, exhibiting a relatively high affinity for the FP (Ki = 83 nM), EP1 (Ki = 95 nM), and EP3 (Ki = 387 nM) receptors. Bimatoprost acid exhibited functional activity at the EP1 (EC50 = 2.7 nM) and FP (EC50 = 2.8-3.8 nM in most cells) receptors. Bimatoprost (nonhydrolyzed amide) also behaved as an FP agonist at the cloned human FP receptor (EC50 = 681 nM), in h-TM (EC50 = 3245 nM) and other cell types. Unoprostone and S-1033 bound with low affinity (Ki = 5.9 microM to > 22 microM) to the FP receptor, were not selective, but activated the FP receptor. In conclusion, travoprost acid has the highest affinity, the highest FP-receptor-selectivity, and the highest potency at the FP receptor as compared to the other ocular hypotensive PG analogs known so far, including free acids of latanoprost, bimatoprost, and unoprostone isopropyl ester.

A key role for prostaglandin I2 in limiting lung mucosal Th2, but not Th1, responses to inhaled allergen.

The cellular events that serve to regulate lung mucosal Th2 responses and limit allergic inflammatory reactions are unclear. Using the DO11.10 TCR transgenic mouse, we developed a model of T cell-mediated pulmonary inflammation and demonstrated that high levels of PGI(2) are produced in the airways following OVA inhalation. Selective inhibition of cyclooxygenase-2 in vivo specifically reduced PGI(2) synthesis and resulted in a marked increase in Th2-mediated, but not Th1-mediated, lung inflammation. The elevated Th2-mediated inflammatory response elicited by the cyclooxygenase-2 inhibitor was associated with enhanced airway hyperreactivity and was coincident with a marked increase in the levels of IL-4, IL-5, and IL-13 in the airways, but a reduction in IL-10 production. In keeping with these observations, we found that the mRNA for the PGI(2) receptor was expressed by Th2, but not Th1, cells, and transcripts for the PGI(2) receptor were induced by IL-4 and OVA peptide stimulation. Interestingly, treatment with PGI(2) or its stable analog, carbaprostacyclin, augmented IL-10 production by Th2 cells. Collectively, our findings reveal a key role for PGI(2) in differentially limiting Th2 responses, possibly by promoting production of the immunosuppressive cytokine IL-10 at the site of allergic lung inflammation. These results indicate an important role for prostanoids generated during inflammation in regulating mucosal T cell responses and highlight a potential risk in the use of cyclooxygenase-2-specific inhibitors by allergic asthmatics.

Fatty acids and immune responses--a new perspective in searching for clues to mechanism.

Dietary essential fatty acids are the precursors for eicosanoids. Among the eicosanoids derived from arachidonic acid, prostaglandin (PG) E2 is known to possess immunosuppressive actions. Thus, it has been a prevailing hypothesis that the immuno-modulatory roles of dietary fatty acids are mediated at least in part through the alteration of PG biosynthesis. PGs exert their biological effects through their cognate receptors. There are four subtypes of PGE receptors (EP1, EP2, EP3, and EP4) so far identified. Although the association of EP receptors with G proteins coupled to adenylate cyclase and the mobilization of intracellular calcium are well documented, downstream signaling pathways for these receptors are virtually unknown. Identification of downstream signaling pathways for each subtype of EP receptors and target genes regulated by the activation of the receptor will help with our understanding of the mechanism by which dietary fatty acids affect immune responses through the modulation of PGE2 biosynthesis. Emerging evidence suggests that fatty acids can additionally act as second messengers, regulators of signal transducing molecules or transcription factors. Acylation with long-chain fatty acids can occur on a variety of signaling molecules and can affect their membrane translocation and functions. Dietary fatty acids can alter functional properties of lipid mediators by changing the composition of acyl moieties of these molecules. Evidence accumulated recently indicates that long-chain unsaturated fatty acids and their metabolites bind and activate peroxisome proliferator-activated receptors (PPARs). PPARs are nuclear hormone receptors and transcription factors that regulate the expression of broad arrays of genes involved not only in lipid and glucose metabolism, but also in immune and inflammatory responses. PPARs may therefore be important cellular targets that mediate modulation of immune responses by dietary fatty acids. Together, it becomes clear now that multiple steps in various receptor-mediated signaling pathways can be modulated by dietary fatty acids. It will be a challenging task to quantitatively determine how different fatty acids alter functional properties of multitude of signaling components and final cellular responses. Elucidating the mechanism of actions of fatty acids on receptor-mediated signaling pathways in immuno-competent cells will provide a new insight for understanding the immuno-modulatory roles of dietary fatty acids.

Tyrosine kinase and c-Jun NH2-terminal kinase mediate hypertrophic responses to prostaglandin F2alpha in cultured neonatal rat ventricular myocytes.

Myocardial infarction results in focal areas of ischemia, hypoxia, necrosis, and decreased contractile function. To compensate for loss of contractile function, remaining viable myocytes undergo hypertrophic growth. Prostaglandin F2alpha (PGF2alpha), which is released from cells of the myocardium during periods of stress such as hypoxia or ischemia/reperfusion, has recently been shown to stimulate hypertrophic growth in neonatal rat ventricular myocytes. In the present study, we determine which growth-related intracellular pathways are required for PGF2alpha to induce morphological and genetic features characteristic of the hypertrophic phenotype. In cardiomyocytes, PGF2alpha increases the hydrolysis of inositol phosphates and induces the translocation of protein kinase C epsilon to the myocyte membrane, consistent with PGF2alpha receptor coupling to Gq. PGF2alpha also activates the extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase pathways. Surprisingly, studies using pharmacological inhibitors and transfection of dominant-interfering proteins demonstrate that PGF2alpha-induced myocyte hypertrophy occurs independent of either PKC, p38, or ERK pathways. Additional studies demonstrate that PGF2alpha stimulates protein tyrosine phosphorylation and activates c-Jun NH2-terminal kinase and suggest that these pathways mediate hypertrophic growth in response to PGF2alpha.

Functional characterization of the ocular prostaglandin f2alpha (PGF2alpha) receptor. Activation by the isoprostane, 12-iso-PGF2alpha.

Prostaglandin F2alpha (PGF2alpha) is a product of cyclooxygenase-catalyzed metabolism of arachidonic acid. Recently, PGF2alpha analogs have been hypothesized to reduce intraocular pressure via relaxation of the ciliary muscle. To investigate the molecular basis of PGF2alpha receptor (FP) activation in the eye, we cloned the FP from a human ciliary body (hcb) cDNA library. The open reading frame of the hcb-FP cDNA was identical to the uterine FP cDNA. The hcb-FP appeared to be predominantly membrane-localized, as visualized by an FP-specific peptide antibody, and coupled to inositol phosphate formation when stably expressed in HEK 293 cells. Interestingly, the hcb-FP could also be activated by the F2 isoprostane, 12-iso-PGF2alpha, in addition to its cognate ligand, PGF2alpha. 12-iso-PGF2alpha was less potent (EC50 = 5 microM) than PGF2alpha (EC50 = 10 nM) in generating inositol phosphates via the hcb-FP in HEK 293 cells. Both ligands also stimulated mitogenesis in NIH 3T3 cells. Although 12-iso-PGF2alpha caused a dose-dependent activation of the FP, it failed to activate the recombinant human prostacyclin receptor and caused only minimal activation of the thromboxane receptor isoforms stably expressed in HEK 293 cells. Four additional F2 isoprostanes, 8-iso-PGF2alpha, IPF2alpha-I, IPF2alpha-III, and 9beta,11beta-PGF2, caused trivial, or no, activation of the FP. Consistent with these observations, only PGF2alpha and 12-iso-PGF2alpha caused rapid homologous desensitization of FP and also exhibited cross-desensitization, with PGF2alpha resulting in a maximum of approximately 60% desensitization. The human FP may thus be activated specifically, by the free radical-catalyzed F2 isoprostane, 12-iso-PGF2alpha, in addition to the cyclooxygenase product, PGF2alpha. Incidental receptor activation by isoprostanes may complement the actions of PGF2alpha in clinical syndromes where oxidant stress and augmented prostaglandin biosynthesis coincide.

Evidence for the distinct nature of F2-isoprostane receptors from those of thromboxane A2.

In rat glomeruli and mesangial cells, the thromboxane A2 (TxA2) mimetic, U-46,619, but not 8-iso-prostaglandin F2alpha (8-iso-PGF2alpha), reduced glomerular inulin space and increased inositol 1,4,5-trisphosphate production, effects abolished by SQ-29,548. In competitive binding studies using 8-iso-[3H]PGF2alpha or [3H]SQ-29,548, mesangial cells displayed TxA2 binding sites but not ones for 8-iso-PGF2alpha. In contrast, rat aortic smooth muscle cells possessed specific binding sites for both TxA2 and 8-iso-PGF2alpha and displayed functional responses to both agonists, such as time- and dose-dependent activation of mitogen-activated protein kinases. In these cells, the mean dissociation constant value for the isoprostane receptor was 31.8 +/- 5.7 nM. When human TxA2 receptor cDNA was expressed in Xenopus oocytes injected with the Ca2+-specific photoprotein, aequorin, 8-iso-PGF2alpha gave much weaker responses than U-46,619. These studies provide the first radioligand binding characteristics of the F2-isoprostane receptor and demonstrate its specific and heterologous cellular localization. These studies support the distinct nature and biological significance of isoprostane receptors and provide a tool for their further molecular characterization.

Antihypertensive effect of thromboxane A2 receptor blockade in genetically hypertensive rats of the Lyon strain.

In order to determine whether the increased renal biosynthesis of thromboxane A2, observed in young genetically hypertensive (LH) rats of the Lyon strain, could be involved in the development of their hypertension, 12 LH female rats were given a specific thromboxane A2 receptor antagonist, AH 23848 (Glaxo Group Research) orally (2 mg/kg twice a day) from 3 to 9 weeks of age. In addition, 12 LH and 12 normotensive (LN) rats were given vehicle only (sodium bicarbonate 8%). The thromboxane receptor antagonist AH 23848, which inhibited platelet aggregation by about 65%, did not modify the renal production of thromboxane A2, prostaglandin I2 (PGI2) or prostaglandin E2 (PGE2). It induced a progressive, potent and long lasting decrease in systolic blood pressure which was normalized in 6-, 7- and 8-week-old LH rats, thus demonstrating the involvement of thromboxane A2 in the onset of hypertension in this model. In contrast with thromboxane synthetase inhibitors, the AH 23848 antihypertensive effect persisted 1 week after the cessation of treatment, showing the superiority of thromboxane A2 receptor blockade over thromboxane synthetase inhibition.

Prostaglandins of the E series inhibit release of noradrenaline in rat hypothalamus by a mechanism unrelated to classical alpha 2 adrenergic presynaptic inhibition.

The effects of eight different prostanoid derivatives (PGs) on the in vitro release of noradrenaline (NA) from rat hypothalamic slices are reported. Prostaglandin E2 (10(-8)-10(-5) M), which does not interfere with the [3H]NA uptake mechanism, inhibited [3H]NA release induced by K+-evoked depolarization. The rank order of inhibition of release of NA for the PGs was: PGE2 greater than PGE1 greater than PGA2 greater than 16, 16-dimethyl-PGE2 greater than 11-epi-PGE2 greater than or equal to 8-iso-PGE2 greater than PGF2 alpha greater than PGD2. It has recently been shown that PGs of the E series specifically bind with a high affinity to membrane preparations of rat hypothalamus. A similar rank order was found for the activity of these PGs in displacing [3H]PGE2 from its binding sites, suggesting that the effect of PGEs on release of NA is mediated by an interaction with PGE2 receptors. Under the same experimental conditions, 10(-6) M clonidine (an alpha 2 adrenoceptor agonist) diminished, and 10(-6) M yohimbine (an alpha adrenoceptor antagonist) increased [3H]NA release, supporting the existence of alpha 2 auto-inhibition. Exposure to 10(-6) M of the alpha 1, alpha 2 adrenergic receptor antagonist phentolamine, a concentration which by itself had no effect on overflow of [3H]NA, blocked the inhibitory effect of clonidine, but failed to antagonize the inhibitory action of PGE2. Moreover, the action of clonidine and yohimbine remained unaffected when PG synthesis was blocked with indomethacin.(ABSTRACT TRUNCATED AT 250 WORDS)