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1.
J Pharmacol Exp Ther ; 373(3): 381-390, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32205366

RESUMO

Prostaglandin (PG) E analogs are used clinically to ripen the cervix and induce labor. However, selective receptor agonists may have potential to improve induction response rates or manage unwanted uterine hypercontractility in conditions such as dysmenorrhea and preterm labor. To characterize their therapeutic value, PGE2 analogs were used to investigate the functional E-type prostanoid (EP) receptor population in isolated human uterus. Responsiveness in mouse tissues was also examined to validate its use as a preclinical model. Uterine samples were obtained from mice at dioestrus (n = 12), term gestation (n = 14), and labor (n = 12) and from the lower uterus of women undergoing hysterectomy (n = 12) or Caesarean section (n = 18). Vehicle and agonist effects were assessed using superfusion and immersion techniques. PGE2 evoked predominant excitatory responses in mouse and relaxation in human tissues. Selective EP4 agonists inhibited tissue activity in both nonpregnant species, while the EP2 mimetic CP533536 also attenuated uterine contractions throughout gestation. The uterotonic effects of the EP3/1 agonist sulprostone were more pronounced than the EP1 agonist ONO-D1-004, corresponding to abundant EP3 receptor expression in all samples. The contractile phenotype in mouse compared with human uteri may relate to regional differences as well as high expression of EP3 receptor transcripts. Similarities in nonpregnant and gestational tissues across species suggest that EP3 may represent a valuable translational drug target for preventing uterine hypercontractility by employing a selective antagonist. SIGNIFICANCE STATEMENT: This research validates the use of nonpregnant mice for preclinical drug discovery of uterine EP receptor targets. To determine the utility of novel drugs and delivery systems at term pregnancy and labor, pharmacological agents interacting with EP3 receptors have clear translational value.


Assuntos
Receptores de Prostaglandina E Subtipo EP2/metabolismo , Reprodução/fisiologia , Útero/metabolismo , Adulto , Animais , Cesárea/métodos , Dinoprostona/análogos & derivados , Dinoprostona/farmacologia , Feminino , Humanos , Camundongos , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Gravidez , Reprodução/efeitos dos fármacos , Contração Uterina/efeitos dos fármacos , Contração Uterina/metabolismo , Útero/efeitos dos fármacos , Adulto Jovem
2.
FASEB J ; 31(1): 368-375, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27770021

RESUMO

The purpose of these studies was to test the hypothesis that a selected polypharmacological approach for treating the prostanoid-mediated component of inflammatory diseases would produce a therapeutic effect superior to global inhibition of prostaglandin (PG) biosynthesis by aspirin-like drugs. The compound studied was AGN 211377, which had been previously shown to produce a superior effect on cytokine release from human macrophages compared with cyclooxygenase (COX) inhibitors. AGN 211377 antagonizes prostanoid prostaglandin D2 (DP)1, DP2, prostaglandin E2 (EP)1, EP4, prostaglandin F2α, and thromboxane A2 receptors but not anti-inflammatory EP2, prostaglandin I2, or EP3 receptors. Established rodent models of ocular inflammatory diseases were used to determine therapeutic effects in living animals. The drugs were administered systemically after predetermination of their blood levels to ensure bioavailability at an appropriate dose level. Whereas compounds selective for a single prostanoid receptor typically exhibited modest but statistically significant inhibition, AGN 211377 profoundly inhibited S-antigen-induced uveitis and laser-induced retinal neovascularization. Consistent with previous polypharmacological studies on chemokine/cytokine release from human macrophages, the prostanoid EP1 receptor played a permissive role in suppressing neovascularization and inflammation in vivo Comparing AGN 211377 with a close structural congener lacking EP1 antagonism (AGN 197727), AGN 197727 was much less active than AGN 211377, but pronounced anti-inflammatory and angiostatic effects were achieved by adding the EP1 antagonist compound (SC-51322) to AGN 197727 in the systemic dosing regimen. Further, AGN 211377 produced superior anti-inflammatory activity compared with the nonsteroidal anti-inflammatory agent ketorolac. These results indicate the value of using a polypharmacological approach in the design of novel therapeutic agents in preference to compounds targeting a single receptor or enzyme. A compound such as AGN 211377 may represent more effective therapy than COX inhibitors in treating uveitis and ocular diseases where neovascularization is a significant part of the pathology.-Woodward, D. F., Wang, J. W., Ni, M., Bauer, A., Martos, J. L., Carling, R. W., Poloso, N. J. In vivo studies validating multitargeting of prostanoid receptors for achieving superior anti-inflammatory effects.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Cinamatos/farmacologia , Neovascularização Patológica/prevenção & controle , Receptores de Prostaglandina/antagonistas & inibidores , Animais , Doenças Autoimunes , Sinalização do Cálcio , Lasers/efeitos adversos , Ratos , Ratos Endogâmicos Lew , Retina/patologia , Retina/efeitos da radiação , Vasos Retinianos/patologia , Vasos Retinianos/efeitos da radiação , Uveíte/tratamento farmacológico , Uveíte/etiologia
3.
Exp Eye Res ; 155: 38-46, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27939447

RESUMO

The goal of the study was to examine secreted protein response and withdrawal profiles from cultured human trabecular meshwork (HTM) cells following short- and long-term glucocorticoid treatment. Primary cultures of five human HTM cell strains isolated from 5 different individual donor eyes were tested. Confluent HTM cells were differentiated in culture media containing 1% FBS for at least one week, and then treated with Dexamethasone (Dex, 100 nM) 3 times/week for 1 or 4 weeks. Cell culture supernatants were collected 3 times per week for 8 weeks. Secretion profiles of myocilin (MYOC), matrix metalloproteinase-2 (MMP2) and fibronectin (FN) were determined by Western blot analysis and MMP2 activity by zymography. Dex treatment reduced MMP2 expression and activity, returning to normal levels shortly after Dex withdrawal in 5 HTM cell strains. All five cell strains significantly upregulated MYOC in response to Dex treatment by an average of 17-fold, but recovery to basal levels after Dex withdrawal took vastly different periods of time depending on cell strain and treatment duration. Dex treatment significantly increased FN secretion in all strains but one, which decreased FN secretion in the presence of Dex. Interestingly, secretion of FN and MYOC negatively correlated during a 4 week recovery period following 4 weeks of Dex treatment. Taken together, the time course and magnitude of response and recovery for three different secreted, extracellular matrix-associated proteins varied greatly between HTM cell strains, which may underlie susceptibility to glucocorticoid-induced ocular hypertension.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Dexametasona/farmacologia , Proteínas da Matriz Extracelular/metabolismo , Proteínas do Olho/metabolismo , Glicoproteínas/metabolismo , Hipertensão Ocular/tratamento farmacológico , Malha Trabecular/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Western Blotting , Células Cultivadas , Glucocorticoides/farmacologia , Humanos , Lactente , Hipertensão Ocular/metabolismo , Hipertensão Ocular/patologia , Doadores de Tecidos , Malha Trabecular/efeitos dos fármacos , Malha Trabecular/metabolismo
4.
FASEB J ; 30(1): 394-404, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26420849

RESUMO

A polypharmacologic approach to prostanoid based anti-inflammatory therapeutics was undertaken in order to exploit both the anti- and proinflammatory properties attributed to the various prostanoid receptors. Multitargeting of selected prostanoid receptors yielded a prototype compound, compound 1 (AGN 211377), that antagonizes prostaglandin D2 receptors (DPs) DP1 (49) and DP2 (558), prostaglandin E2 receptors (EPs) EP1 (266) and EP4 (117), prostaglandin F2α receptor (FP) (61), and thromboxane A2 receptor (TP) (11) while sparing EP2, EP3, and prostaglandin I2 receptors (IPs); Kb values (in nanomoles) are given in parentheses. Compound 1 evoked a pronounced inhibition of cytokine/chemokine secretion from lipopolysaccharide or TNF-α stimulated primary human macrophages. These cytokine/chemokines included cluster of designation 40 receptor (CD40), epithelial-derived neutrophil-activating protein 78 (ENA-78), granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), IL-8, IL-18, monocyte chemotactic protein-1 (CCL2) (MCP-1), tissue plasminogen activator inhibitor (PAI-1), and regulated on activation, normal T cell expressed and secreted (RANTES). In contrast, the inhibitory effects of most antagonists selective for a single receptor were modest or absent, and selective EP2 receptor blockade increased cytokine release in some instances. Compound 1 also showed clear superiority to the cyclooxygenase inhibitors diclofenac and rofecoxib. These findings reveal that blockade of multiple prostanoid receptors, with absent antagonism of EP2 and IP, may provide more effective anti-inflammatory activity than global suppression of prostanoid synthesis or highly selective prostanoid receptor blockade. These investigations demonstrate the first working example of prostanoid receptor polypharmacology for potentially safer and more effective anti-inflammatory therapeutics by blocking multiple proinflammatory receptors while sparing those with anti-inflammatory activity.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Cinamatos/farmacologia , Macrófagos/efeitos dos fármacos , Receptores de Prostaglandina/antagonistas & inibidores , Receptores de Tromboxanos/antagonistas & inibidores , Anti-Inflamatórios não Esteroides/síntese química , Compostos Bicíclicos Heterocíclicos com Pontes/síntese química , Células Cultivadas , Cinamatos/síntese química , Humanos , Macrófagos/metabolismo , Especificidade por Substrato
5.
J Pharmacol Exp Ther ; 358(2): 173-80, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27217589

RESUMO

Prostaglandin E2 (PGE2)-2-glyceryl ester is a cyclo-oxygenase 2 product of the endocannabinoid 2-arachidonyl glycerol. It is claimed as pharmacologically novel, but this is complicated by rapid and irreversible isomerization to the 1(3) ester. For ocular studies, enzymatic hydrolysis of the ester moiety creates an additional complication. PG-glyceryl esters were stabilized to isomerization and hydrolysis by replacing the noncarbonyl O with NH, to form the serinolamide and propanediolamide as stable analogs of PG-2-glyceryl and PG-2-1(3) glyceryl esters, respectively. Intraocular pressure was measured in conscious dogs and conscious laser-induced ocular hypertensive monkeys. Pharmacological studies involved stable transfectants for each of the human recombinant prostanoid receptors and the isolated feline iris for prostamide activity. PGE2-serinolamide and PGE2- propanediolamide were essentially inactive at all receptors except the EP3 receptor (EC50, ∼500 nM). This obliged elucidation of EP3 receptor involvement in the intraocular pressure response to these PGE2-glycyerl ester analogs. Since the EP3 receptor agonists sulprostone and GR 63799 did not lower monkey intraocular pressure, a role for EP3 receptors in mediating the effects of PGE2-serinolamide and PGE2-propanediolamide is not indicated. PGE2-glyceryl ester (0.01% and 0.1%) substantially lowered intraocular pressure in monkeys. PGE2-propanediolamide was more efficacious than PGE2-serinolamide in lowering intraocular pressure in monkey eyes, but both appeared equieffective in dog eyes. PGE2-serinolamide dose-dependently (0.01- 0.1%) lowered intraocular pressure in both species, but PGF2 α-serinolamide was inactive. In conclusion, stable PGE2-glyceryl ester analogs lowered intraocular pressure. These findings are consistent with the presence of a PGE2-glyceryl ester-specific recognition site in the eye.


Assuntos
Dinoprostona/análogos & derivados , Pressão Intraocular/efeitos dos fármacos , Animais , Gatos , Dinoprostona/farmacologia , Cães , Feminino , Iris/efeitos dos fármacos , Iris/fisiologia , Macaca fascicularis , Masculino , Contração Muscular/efeitos dos fármacos
6.
J Lipid Res ; 56(8): 1419-33, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26031663

RESUMO

Arachidonoyl ethanolamine (anandamide) and pros-taglandin ethanolamines (prostamides) are biologically active derivatives of arachidonic acid. Although available through different precursor phospholipids, there is considerable overlap between the biosynthetic pathways of arachidonic acid-derived eicosanoids and anandamide-derived prostamides. Prostamides exhibit physiological actions and are involved in ocular hypotension, smooth muscle contraction, and inflammatory pain. Although topical application of bimatoprost, a structural analog of prostaglandin F2α ethanolamide (PGF2α-EA), is currently a first-line treatment for ocular hypertension, the endogenous production of prostamides and their biochemical precursors in corneal tissue has not yet been reported. In this study, we report the presence of anandamide, palmitoyl-, stearoyl-, α-linolenoyl docosahexaenoyl-, linoleoyl-, and oleoyl-ethanolamines in rabbit cornea, and following treatment with anandamide, the formation of PGF2α-EA, PGE2-EA, PGD2-EA by corneal extracts (all analyzed by LC/ESI-MS/MS). A number of N-acyl phosphatidylethanolamines, precursors of anandamide and other fatty acyl ethanolamines, were also identified in corneal lipid extracts using ESI-MS/MS. These findings suggest that the prostamide and fatty acid ethanolamine pathways are operational in the cornea and may provide valuable insight into corneal physiology and their potential influence on adjacent tissues and the aqueous humor.


Assuntos
Córnea/metabolismo , Dinoprostona/biossíntese , Dinoprostona/metabolismo , Etanolaminas/metabolismo , Animais , Dinoprostona/química , Etanolaminas/química , Coelhos
7.
J Biol Chem ; 288(32): 23307-21, 2013 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-23801328

RESUMO

Lipid mediators variedly affect adipocyte differentiation. Anandamide stimulates adipogenesis via CB1 receptors and peroxisome proliferator-activated receptor γ. Anandamide may be converted by PTGS2 (COX2) and prostaglandin F synthases, such as prostamide/prostaglandin F synthase, to prostaglandin F2α ethanolamide (PGF2αEA), of which bimatoprost is a potent synthetic analog. PGF2αEA/bimatoprost act via prostaglandin F2αFP receptor/FP alt4 splicing variant heterodimers. We investigated whether prostamide signaling occurs in preadipocytes and controls adipogenesis. Exposure of mouse 3T3-L1 or human preadipocytes to PGF2αEA/bimatoprost during early differentiation inhibits adipogenesis. PGF2αEA is produced from anandamide in preadipocytes and much less so in differentiating adipocytes, which express much less PTGS2, FP, and its alt4 splicing variant. Selective antagonism of PGF2αEA receptors counteracts prostamide effects on adipogenesis, as does inhibition of ERK1/2 phosphorylation. Selective inhibition of PGF2αEA versus prostaglandin F2α biosynthesis accelerates adipogenesis. PGF2αEA levels are reduced in the white adipose tissue of high fat diet-fed mice where there is a high requirement for new adipocytes. Prostamides also inhibit zebrafish larval adipogenesis in vivo. We propose that prostamide signaling in preadipocytes is a novel anandamide-derived antiadipogenic mechanism.


Assuntos
Adipócitos/metabolismo , Adipogenia/fisiologia , Ácidos Araquidônicos/metabolismo , Dinoprostona/análogos & derivados , Endocanabinoides/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Alcamidas Poli-Insaturadas/metabolismo , Células 3T3-L1 , Adipócitos/citologia , Animais , Ácidos Araquidônicos/genética , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/biossíntese , Dinoprostona/genética , Endocanabinoides/genética , Feminino , Humanos , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação/fisiologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
8.
FASEB J ; 27(2): 557-67, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23104985

RESUMO

Balding causes widespread psychological distress but is poorly controlled. The commonest treatment, minoxidil, was originally an antihypertensive drug that promoted unwanted hair. We hypothesized that another serendipitous discovery, increased eyelash growth side-effects of prostamide F(2α)-related eyedrops for glaucoma, may be relevant for scalp alopecias. Eyelash hairs and follicles are highly specialized and remain unaffected by androgens that inhibit scalp follicles and stimulate many others. Therefore, we investigated whether non-eyelash follicles could respond to bimatoprost, a prostamide F(2α) analog recently licensed for eyelash hypotrichosis. Bimatoprost, at pharmacologically selective concentrations, increased hair synthesis in scalp follicle organ culture and advanced mouse pelage hair regrowth in vivo compared to vehicle alone. A prostamide receptor antagonist blocked isolated follicle growth, confirming a direct, receptor-mediated mechanism within follicles; RT-PCR analysis identified 3 relevant receptor genes in scalp follicles in vivo. Receptors were located in the key follicle regulator, the dermal papilla, by analyzing individual follicular structures and immunohistochemistry. Thus, bimatoprost stimulates human scalp follicles in culture and rodent pelage follicles in vivo, mirroring eyelash behavior, and scalp follicles contain bimatoprost-sensitive prostamide receptors in vivo. This highlights a new follicular signaling system and confirms that bimatoprost offers a novel, low-risk therapeutic approach for scalp alopecias.


Assuntos
Alopecia/tratamento farmacológico , Amidas/uso terapêutico , Cloprostenol/análogos & derivados , Administração Tópica , Adulto , Alopecia/genética , Alopecia/metabolismo , Amidas/administração & dosagem , Animais , Anti-Hipertensivos/administração & dosagem , Anti-Hipertensivos/uso terapêutico , Sequência de Bases , Bimatoprost , Cloprostenol/administração & dosagem , Cloprostenol/uso terapêutico , Feminino , Glaucoma/tratamento farmacológico , Folículo Piloso/efeitos dos fármacos , Folículo Piloso/crescimento & desenvolvimento , Folículo Piloso/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Pessoa de Meia-Idade , Técnicas de Cultura de Órgãos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Prostaglandina/antagonistas & inibidores , Receptores de Prostaglandina/efeitos dos fármacos , Receptores de Prostaglandina/genética , Receptores de Prostaglandina/metabolismo , Couro Cabeludo/efeitos dos fármacos , Couro Cabeludo/metabolismo , Adulto Jovem
9.
J Mol Endocrinol ; 73(1)2024 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-38639976

RESUMO

The prostanoid G protein-coupled receptor (GPCR) EP2 is widely expressed and implicated in endometriosis, osteoporosis, obesity, pre-term labour and cancer. Internalisation and intracellular trafficking are critical for shaping GPCR activity, yet little is known regarding the spatial programming of EP2 signalling and whether this can be exploited pharmacologically. Using three EP2-selective ligands that favour activation of different EP2 pathways, we show that EP2 undergoes limited agonist-driven internalisation but is constitutively internalised via dynamin-dependent, ß-arrestin-independent pathways. EP2 was constitutively trafficked to early and very early endosomes (VEE), which was not altered by ligand activation. APPL1, a key adaptor and regulatory protein of the VEE, did not impact EP2 agonist-mediated cAMP. Internalisation was required for ~70% of the acute butaprost- and AH13205-mediated cAMP signalling, yet PGN9856i, a Gαs-biased agonist, was less dependent on receptor internalisation for its cAMP signalling, particularly in human term pregnant myometrial cells that endogenously express EP2. Inhibition of EP2 internalisation partially reduced calcium signalling activated by butaprost or AH13205 and had no effect on PGE2 secretion. This indicates an agonist-dependent differential spatial requirement for Gαs and Gαq/11 signalling and a role for plasma membrane-initiated Gαq/11-Ca2+-mediated PGE2 secretion. These findings reveal a key role for EP2 constitutive internalisation in its signalling and potential spatial bias in mediating its downstream functions. This, in turn, could highlight important considerations for future selective targeting of EP2 signalling pathways.


Assuntos
Proteínas de Ligação ao GTP , Receptores de Prostaglandina E Subtipo EP2 , Transdução de Sinais , Feminino , Humanos , Gravidez , Alprostadil/análogos & derivados , Alprostadil/farmacologia , Alprostadil/metabolismo , AMP Cíclico/metabolismo , Endossomos/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Miométrio/metabolismo , Transporte Proteico , Receptores de Prostaglandina E Subtipo EP2/metabolismo
10.
Exp Dermatol ; 22(2): 145-8, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23278986

RESUMO

Studies on bimatoprost were performed with two objectives: (i) to determine whether bimatoprost possesses hair growth-stimulating properties beyond eyelash hypertrichosis and (ii) to investigate the biodisposition of bimatoprost in skin for the first time. Bimatoprost, at the dose used clinically for eyelash growth (0.03%) and given once daily for 14 days, increased pelage hair growth in C57/black 6 mice. This occurred as a much earlier onset of new hair growth in shaved mice and the time taken to achieve complete hair regrowth, according to photographic documentation and visual assessment. Bimatoprost biodisposition in the skin was determined at three concentrations: 0.01%, 0.03% and 0.06%. Dose-dependent C(max) values were obtained (3.41, 6.74, 12.3 µg/g tissue), and cutaneous bimatoprost was well maintained for 24 h following a single dose. Bimatoprost was recovered from the skin only as the intact molecule, with no detectable levels of metabolites. Thus, bimatoprost produces hypertrichosis as the intact molecule.


Assuntos
Amidas/farmacologia , Amidas/farmacocinética , Cloprostenol/análogos & derivados , Hipertricose/induzido quimicamente , Pele/efeitos dos fármacos , Animais , Anti-Hipertensivos/farmacologia , Bimatoprost , Cloprostenol/farmacocinética , Cloprostenol/farmacologia , Relação Dose-Resposta a Droga , Pestanas , Cabelo/fisiologia , Hipertricose/patologia , Camundongos , Camundongos Endogâmicos C57BL , Prostaglandinas/metabolismo , Pele/patologia , Fatores de Tempo
11.
Artigo em Inglês | MEDLINE | ID: mdl-36399889

RESUMO

Bimatoprost is a synthetic prostamide F2α analog that down-regulates adipogenesis in vitro. This effect has been attributed to participation in a negative feedback loop that regulates anandamide-induced adipogenesis. A follow-on investigation has now been conducted into the broader metabolic effects of bimatoprost using rats under both normal state and obesity-inducing conditions. Chronic bimatoprost administration attenuated weight gain in a dose dependent-manner in rats fed either standard [max effect -7%] or obesity-promoting diets [max effect -23%] over a 9-10 week period. Consistent with these findings, bimatoprost promoted satiety as measured by decreased food intake [max effect, -7%], gastric emptying [max effect, -33-50%] and decreased circulating concentrations of the gut hormones, ghrelin and GLP-1 [max effect, -33-50%]. Additionally, subcutaneous, and visceral fat mass were distinctly affected by treatment [-30% diet independent]. Taken together, these results suggest that bimatoprost regulates energy homeostasis through promoting satiety and a decrease in food intake. These newly reported activities of bimatoprost reveal an additional method of metabolic disease intervention for potential therapeutic exploitation.


Assuntos
Obesidade , Aumento de Peso , Ratos , Animais , Bimatoprost , Obesidade/tratamento farmacológico , Dieta , Adipogenia
12.
Cell Rep ; 40(10): 111318, 2022 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-36070698

RESUMO

Current strategies to manage preterm labor center around inhibition of uterine myometrial contractions, yet do not improve neonatal outcomes as they do not address activation of inflammation. Here, we identify that during human labor, activated oxytocin receptor (OTR) reprograms the prostaglandin E2 receptor, EP2, in the pregnant myometrium to suppress relaxatory/Gαs-cAMP signaling and promote pro-labor/inflammatory responses via altered coupling of EP2 from Gαq/11 to Gαi/o. The ability of EP2 to signal via Gαi/o is recapitulated with in vitro OT and only following OTR activation, suggesting direct EP2-OTR crosstalk. Super-resolution imaging with computational modeling reveals OT-dependent reorganization of EP2-OTR complexes to favor conformations for Gαi over Gαs activation. A selective EP2 ligand, PGN9856i, activates the relaxatory/Gαs-cAMP pathway but not the pro-labor/inflammatory responses in term-pregnant myometrium, even following OT. Our study reveals a mechanism, and provides a potential therapeutic solution, whereby EP2-OTR functional associations could be exploited to delay preterm labor.


Assuntos
Trabalho de Parto , Trabalho de Parto Prematuro , Feminino , Humanos , Recém-Nascido , Trabalho de Parto/metabolismo , Miométrio/metabolismo , Gravidez , Receptores de Ocitocina , Contração Uterina/fisiologia
13.
Br J Ophthalmol ; 105(11): 1610-1616, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-33239414

RESUMO

BACKGROUND: A single application of JV-GL1 substantially lowers non-human primate intraocular pressure (IOP) for about a week, independent of dose. This highly protracted effect does not correlate with its ocular biodisposition or correlate with the once-daily dosing regimen for other prostanoid EP2 receptor agonists such as trapenepag or omidenepag. The underlying pharmacological mechanism for the multiday extended activity of JV-GL1 is highly intriguing. The present studies were intended to determine EP2 receptor involvement in mediating the long-term ocular hypotensive activity of JV-GL1 by using mice genetically deficient in EP2 receptors. METHODS: The protracted IOP reduction produced by JV-GL1 was investigated in C57BL/6J and EP2 receptor knock-out mice (B6.129-Ptger2tm1Brey /J; EP2KO). Both ocular normotensive and steroid-induced ocular hypertensive (SI-OHT) mice were studied. IOP was measured tonometrically under general anaesthesia. Aqueous humour outflow facility was measured ex vivo using iPerfusion in normotensive C57BL/6J mouse eyes perfused with 100 nM de-esterified JV-GL1 and in SI-OHT C57BL/6J mouse eyes that had received topical JV-GL1 (0.01%) 3 days prior. RESULTS: Both the initial 1-day and the protracted multiday effects of JV-GL1 in the SI-OHT model for glaucoma were abolished by deletion of the gene encoding the EP2 receptor. Thus, JV-GL1 did not lower IOP in SI-OHT EP2KO mice, but in littermate SI-OHT EP2WT control mice, JV-GL1 statistically significantly lowered IOP for 4-6 days. CONCLUSIONS: Both the 1-day and the long-term effects of JV-GL1 on IOP are entirely EP2 receptor dependent.


Assuntos
Pressão Intraocular , Hipertensão Ocular , Hipotensão Ocular , Animais , Anti-Hipertensivos/uso terapêutico , Pressão Intraocular/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Hipertensão Ocular/tratamento farmacológico , Hipotensão Ocular/tratamento farmacológico , Soluções Oftálmicas/administração & dosagem , Tonometria Ocular
14.
Artigo em Inglês | MEDLINE | ID: mdl-33418484

RESUMO

Prostamide/prostaglandin F synthase (PM/PGFS) is an enzyme with very narrow substrate specificity and is dedicated to the biosynthesis of prostamide F2α and prostaglandin F2α (PGF2α.). The importance of this enzyme, relative to the aldo-keto reductase (AKR) series, in providing functional tissue prostamide F2α levels was determined by creating a line of PM/PGFS gene deleted mice. Deletion of the gene encoding PM/PGFS (Fam213b / Prxl2b) was accomplished by a two exon disruption. Prostamide F2α levels in wild type (WT) and PM/PGFS knock-out (KO) mice were determined by LC/MS/MS. Deletion of Fam213b (Prxl2b) had no observed effect on behavior, appetite, or fertility. In contrast, tonometrically measured intraocular pressure was significantly elevated by approximately 4 mmHg in PM/PGFS KO mice compared to littermate WT mice. Outflow facility was measured in enucleated mouse eyes using the iPerfusion system. No effect on pressure dependent outflow facility occurred, which is consistent with the effects of prostamide F2α and PGF2α increasing outflow through the unconventional pathway. The elevation of intraocular pressure caused by deletion of the gene encoding the PM/PGFS enzyme likely results from a diversion of the endoperoxide precursor pathway to provide increased levels of those prostanoids known to raise intraocular pressure, namely prostaglandin D2 (PGD2) and thromboxane A2 (TxA2). It follows that PM/PGFS may serve an important regulatory role in the eye by providing PGF2α and prostamide F2α to constrain the influence of those prostanoids that raise intraocular pressure.


Assuntos
Dinoprosta/metabolismo , Dinoprostona/análogos & derivados , Deleção de Genes , Hidroxiprostaglandina Desidrogenases/metabolismo , Animais , Cromatografia Líquida , Dinoprostona/metabolismo , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Hidroxiprostaglandina Desidrogenases/genética , Pressão Intraocular , Masculino , Camundongos , Espectrometria de Massas em Tandem , Tonometria Ocular
15.
Mol Pharmacol ; 77(6): 1025-36, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20335389

RESUMO

The EP1 prostanoid receptor is one of four subtypes whose cognate physiological ligand is prostaglandin-E2 (PGE(2)). It is in the family of G-protein-coupled receptors and is known to activate Ca(2+) signaling, although relatively little is known about other aspects of E-type prostanoid receptor (EP) 1 receptor signaling. In human embryonic kidney (HEK) cells expressing human EP1 receptors, we now show that PGE(2) stimulation of the EP1 receptor up-regulates the expression of hypoxia-inducible factor-1 alpha (HIF-1 alpha), which can be completely blocked by pertussis toxin, indicating coupling to G(i/o). This up-regulation of HIF-1 alpha occurs under normoxic conditions and could be inhibited with wortmannin, Akt inhibitor, and rapamycin, consistent with the activation of a phosphoinositide-3 kinase/Akt/mammalian target of rapamycin (mTOR) signaling pathway, respectively. In contrast to the hypoxia-induced up-regulation of HIF-1 alpha, which involves decreased protein degradation, the up-regulation of HIF-1 alpha by the EP1 receptor was associated with the phosphorylation of ribosomal protein S6 (rpS6), suggesting activation of the ribosomal S6 kinases and increased translation. Stimulation of endogenous EP1 receptors in human HepG2 hepatocellular carcinoma cells recapitulated the normoxic up-regulation of HIF-1 alpha observed in HEK cells, was sensitive to pertussis toxin, and involved the activation of mTOR signaling and phosphorylation of rpS6. In addition, treatment of HepG2 cells with sulprostone, an EP1-selective agonist, up-regulated the mRNA expression of vascular endothelial growth factor-C, a HIF-regulated gene. HIF-1 alpha is known to promote tumor growth and metastasis and is often up-regulated in cancer. Our findings provide a potential mechanism by which increased PGE(2) biosynthesis could up-regulate the expression of HIF-1 alpha and promote tumorigenesis.


Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Receptores de Prostaglandina E/metabolismo , Transdução de Sinais , Regulação para Cima , Androstadienos/farmacologia , Linhagem Celular , Dinoprostona/farmacologia , Genes Reporter , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fosfatidilinositol 3-Quinases/farmacologia , Fosforilação , Reação em Cadeia da Polimerase , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Prostaglandina E/agonistas , Receptores de Prostaglandina E Subtipo EP1 , Sirolimo/farmacologia , Serina-Treonina Quinases TOR , Regulação para Cima/efeitos dos fármacos , Wortmanina
16.
Prostaglandins Other Lipid Mediat ; 92(1-4): 19-24, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20152925

RESUMO

2-Arachidonoylglycerol is oxygenated by cyclooxygenase-2 to form prostaglandin glyceryl esters. Previous work in this laboratory has suggested that PGE(2)-G activates a novel G protein-coupled receptor in a murine macrophage-like cell line, RAW 264.7. To probe the structural determinants for the putative receptor in RAW 264.7 cells, a panel of 10 analogs was tested for their ability to increase intracellular calcium. These analogs included PGE(2)- and PGF(2alpha)-ethanolamide, 4 PGE(2) glyceryl ester analogs, and 4 PGF(2alpha) glyceryl ester analogs. The glyceryl ester analogs differed in the positioning of the hydroxyl groups in the glycerol moiety and the type of linker (ester, amide, or thioester) of the prostaglandin to the glycerol moiety. Compounds were also evaluated in a human non-small cell lung cancer cell line (H1819). The glycerol moiety was required for the calcium response. All glyceryl ester analogs but not ethanolamides caused a concentration-dependent increase in calcium levels in both RAW 264.7 and H1819 cells. An amide or ester linkage was preferable to a thioester linkage. The EC(50) values did not significantly change when the positioning of the hydroxyls was varied. This calcium response induced by the glyceryl ester analogs appears to be independent of the putative hydrolysis products, PGE(2) and PGF(2alpha), and appears to represent a novel signaling pathway.


Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Dinoprosta/química , Dinoprosta/farmacologia , Dinoprostona/química , Dinoprostona/farmacologia , Ésteres/química , Animais , Linhagem Celular Tumoral , Etanolamina/química , Humanos , Camundongos , Relação Estrutura-Atividade
17.
J Ocul Pharmacol Ther ; 36(8): 636-648, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32640926

RESUMO

Purpose: JV-GL1 is an efficacious, potent, and long-acting antiglaucoma agent, according to studies in ocular normotensive and hypertensive monkeys. As an obligatory step in the drug development process, studies with exaggerated doses and an accelerated dosing schedule for JV-GL1 were performed in a second species (dog). Methods: Intraocular pressure (IOP) was measured by pneumatonometry in conscious Beagle dogs, which remained conscious throughout the study and gently restrained by hand. Pupil diameter was measured with an Optistick. Ocular surface hyperemia was visually assessed and scored according to a 1-3 assessment scale. Results: JV-GL1, as a 0.01% eye drop, produced significantly greater reductions in IOP than the original clinical dose of bimatoprost (0.03%). JV-GL1 and its free acid enzymatic hydrolysis product PGN 9856, over a 0.01%-0.1% dose range, reduced IOP to ≤10 mm Hg. JV-GL1 and PGN 9856 produced no miosis but a similar degree of ocular surface hyperemia to bimatoprost. Although PGN 9862, a close congener of PGN 9856, was very active as the free acid, esterification essentially abolished its ocular hypotensive activity and ocular surface redness. Conclusion: JV-GL1 was confirmed as a highly effective and potent ocular hypotensive, exceeding the activity of bimatoprost. A similar degree of ocular surface redness was apparent for both compounds, given as eye drops, but no other effects occurred. Results with PGN 9862 and its isopropyl ester confirmed that PGN 9862-isopropyl ester is not bioavailable in the eye and not susceptible to enzymatic hydrolysis in ocular tissues, a first for C1 ester prodrugs in the eye.


Assuntos
Acetatos/farmacologia , Anti-Hipertensivos/farmacologia , Compostos de Bifenilo/farmacologia , Olho/efeitos dos fármacos , Glaucoma/tratamento farmacológico , Acetatos/administração & dosagem , Acetatos/efeitos adversos , Acetatos/química , Administração Tópica , Animais , Anti-Hipertensivos/administração & dosagem , Anti-Hipertensivos/efeitos adversos , Anti-Hipertensivos/química , Bimatoprost/farmacologia , Disponibilidade Biológica , Compostos de Bifenilo/administração & dosagem , Compostos de Bifenilo/efeitos adversos , Compostos de Bifenilo/química , Cães , Desenvolvimento de Medicamentos/métodos , Olho/metabolismo , Olho/fisiopatologia , Feminino , Hidrólise , Hiperemia/induzido quimicamente , Pressão Intraocular/efeitos dos fármacos , Masculino , Modelos Animais , Soluções Oftálmicas/administração & dosagem , Segurança
18.
ACS Pharmacol Transl Sci ; 3(6): 1199-1210, 2020 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-33344897

RESUMO

It has previously been reported that a prototypical compound (AGN 211377), which blocks pro-inflammatory prostanoid receptors (DP1, DP2, EP1, EP4, FP, TP) and leaves open IP and EP2 receptors so that their anti-inflammatory properties could be exerted, produced superior inhibitory effects on cytokine release from human macrophages compared to cyclooxygenase (COX) inhibitors. This favorable activity profile translated into animal studies, with AGN 211377 exceeding the level of inhibition afforded by COX inhibition. AGN 211377 was not, however, a practical drug candidate, having poor bioavailability and cost of goods concerns. Compound 1 (designated AGN 225660) represents a second-generation compound with an entirely different "druggable" core structure. Such a dramatic change in chemical scaffold created uncertainty with respect to matching the effects of AGN 211377. AGN 225660 inhibited RANTES, IL-8, and MCP-1 secretion by at least 50%, from TNFα activated human macrophages. Although AGN 225660 reduced TNFα-evoked MCP-1 release from human monocyte-derived macrophages, it increased LPS-induced MCP-1 secretion (up to 2-fold) from human monocyte-derived dendritic cells. However, AGN 225660 inhibited the release of IL12p 70 and IL-23 from human monocyte-derived dendritic cells stimulated by LPS by more than 70%. This effect of AGN 225660 was reproduced in part by the prototype compound AGN 211377 and a combination of selective DP1, EP1, EP4, FP, and TP antagonists. These findings suggest important effects on T cell skewing and disease modification by this class of therapeutic agents. AGN 225660 exhibited good ocular bioavailability and was active in reducing ocular inflammation associated with phacoemulsification surgery, LPS, and arachidonic acid induced uveitis.

20.
J Ocul Pharmacol Ther ; 35(2): 98-105, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30547709

RESUMO

PURPOSE: The purpose of these studies was (1) to investigate the ability of human M1 phenotype macrophages to secrete vascular endothelial growth factor (VEGF) and the influence of prostacyclin receptor (IP) stimulation (2) to evaluate the contribution of the proangiogenic prostanoid prostacyclin to experimental choroidal neovascularization Methods: Human macrophages derived from primary blood mononuclear cells were functionally biased toward the M1 phenotype by using tumor necrosis factor α (TNFα). Experimental choroidal neovascularization was produced by laser photocoagulation. Antagonist drugs RO-3244794 (IP antagonist) and GW 627368 (EP4 antagonist) were administered according to an optimal dosing regimen that was predetermined by bioavailability studies. RESULTS: IP receptor stimulation had diametrically opposed effects on VEGF release compared with reported data on cytokine/chemokine secretion from human macrophages. For example, the IP agonist cicaprost stimulated VEGF secretion although it inhibits monocyte chemoattractant protein-1 (MCP-1) secretion: both would favor a proangiogenic effect. The IP receptor antagonist RO-3244794 produced an ∼20% statistically significant reduction in the neovascularized lesion area in the choroidal neovascularization model, which was a similar level to that produced by the EP4 antagonist GW 627368. Combining the 2 drugs produced a statistically significant reduction in neovascularization but only of slightly greater magnitude than that obtained with each antagonist administered alone. CONCLUSIONS: IP receptor stimulation potently and highly efficaciously promoted VEGF release from human M1 macrophages, indicating a possible contribution of the M1 macrophage subtype to VEGF-induced choroidal neovascularization. Studies in living animals suggest that prostacyclin and its target IP receptor contribute to choroidal neovascularization, although to a more modest extent than might have been expected.


Assuntos
Anti-Hipertensivos/farmacologia , Neovascularização de Coroide/tratamento farmacológico , Epoprostenol/farmacologia , Macrófagos/efeitos dos fármacos , Soluções Oftálmicas/farmacologia , Animais , Células Cultivadas , Quimiocina CCL2/análise , Quimiocina CCL2/deficiência , Quimiocina CCL2/metabolismo , Neovascularização de Coroide/metabolismo , Neovascularização de Coroide/patologia , Modelos Animais de Doenças , Humanos , Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Camundongos Knockout , Ratos , Ratos Sprague-Dawley , Fatores de Crescimento do Endotélio Vascular/análise , Fatores de Crescimento do Endotélio Vascular/metabolismo
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