No abstract

We investigated whether prostaglandin ethanolamides (prostamides) E 2 , F 2␣ , and D 2 exert some of their effects by 1) activating prostanoid receptors either per se or after conversion into the corresponding prostaglandins; 2) interacting with proteins for the inactivation of the endocannabinoid N-arachidonoylethanolamide (AEA), for example fatty acid amide hydrolase (FAAH), thereby enhancing AEA endogenous levels; or 3) activating the vanilloid receptor type-1 (TRPV1). Prostamides potently stimulated cat iris contraction with potency approaching that of the corresponding prostaglandins. However, prostamides D 2 , E 2 , and F 2␣ exhibited no meaningful interaction with the cat recombinant FP receptor, nor with human recombinant DP, EP 1-4 , FP, IP, and TP prostanoid receptors. Prostamide F 2␣ was also very weak or inactive in a panel of bioassays specific for the various prostanoid receptors. None of the prostamides inhibited AEA enzymatic hydrolysis by FAAH in cell homogenates, or AEA cellular uptake in intact cells. Furthermore, less than 3% of the compounds were hydrolyzed to the corresponding prostaglandins when incubated for 4 h with homogenates of rat brain, lung, or liver, and cat iris or ciliary body. Very little temperature-dependent uptake of prostamides was observed after incubation with rat brain synaptosomes or RBL-2H3 cells. We suggest that prostamides' most prominent pharmacological actions are not due to transformation into prostaglandins, activation of prostanoid receptors, enhancement of AEA levels, or gating of TRPV1 receptors, but possibly to interaction with novel receptors that seem to be functional in the cat iris.

Replacement of the carboxylic acid group of prostaglandin (PG) F 2␣ with a nonacidic moiety, such as hydroxyl, methoxy, or amido, results in compounds with unique pharmacology. Bimatoprost (AGN 192024) is also a pharmacologically novel PGF 2␣ analog, where the carboxylic acid is replaced by a neutral ethylamide substituent. Bimatoprost potently contracted the feline lung parenchymal preparation (EC 50 value of 35-55 nM) but exhibited no meaningful activity in a variety of PG-sensitive tissue and cell preparations. Its activity seemed unrelated to FP receptor stimulation according to the following evidence. 1) Bimatoprost exhibited no meaningful activity in tissues and cells containing functional FP receptors. 2) Bimatoprost activity in the cat lung parenchyma is not species-specific because its potent activity in this preparation could not be reproduced in cells stably expressing the feline FP receptor. 3) Radioligand binding studies using feline and human recombinant FP receptors exhibited minimal competition versus [3 H]17-phenyl PGF 2a for Bimatoprost. 4) Bimatoprost pretreatment did not attenuate PGF 2␣ -induced Ca 2ϩ signals in Swiss 3T3 cells. 5) Regional differences were apparent for Bimatoprost but not FP agonist effects in the cat lung. Bimatoprost reduced intraocular pressure in ocular normotensive and hypertensive monkeys over a 0.001 to 0.1% dose range. A single-dose and multiple-dose ocular distribution/metabolism studies using [ 3 H]Bimatoprost (0.1%) were performed. Within the globe, bimatoprost concentrations were 10-to 100-fold higher in anterior segment tissues compared with the aqueous humor. Bimatoprost was overwhelmingly the predominant molecular species identified at all time points in ocular tissues, indicating that the intact molecule reduces intraocular pressure.Eicosanoids and related fatty acids have long been the subject of extensive investigation. More recently, it has become apparent that the corresponding neutral lipids exist for several fatty acids (Devane et al

1 The polymerase chain reaction (PCR) was used in combination with plaque hybridization analysis to clone four variants of the EP3 prostaglandin receptor from a human small intestine cDNA library. 2 Three of these variants, i.e. the EP3A, EP3E and EP3D, share the same primary amino acid sequence except for their carboxyl termini, which diverge from one another at the same point, approximately 10 amino acids away from the end of the seventh membrane spanning domain of the receptor. The fourth variant (EP3A1) has a nucleotide coding sequence identical to EP3A but has a completely different 3' untranslated sequence. 3 The carboxyl termini of the three isoforms differ most obviously in length with the EP3A being the longest (41 amino acids) and the EP3E being the shortest (16 amino acids). They also differ in content with the EP3A containing 9 serine and threonines in its carboxyl terminus and the EP3E none. 4 Transient expression in eukaryotic cells showed that the human EP3 receptor variants had similar but not identical radioligand binding properties and differed in their functional coupling to second messenger pathways. Up to 3 pmol mg-' protein of [3H]-prostaglandin E2 binding could be obtained with more than 95% specific binding. Using a reporter gene assay, as a measure of intracellular cyclic AMP levels, the EP3A coupled more efficiently to the inhibition of adenylyl cyclase than did the EP3E. 5 PCR was used to confirm the presence of mRNAs encoding the four human EP3 receptor variants in tissues of the human small intestine, heart and pancreas. These findings indicate that the EP3 receptor variants identified here are likely to be expressed in tissues. The differences in the carboxyl termini at the protein level, and in the 3' untranslated regions at the mRNA level, could be profound in terms of the regulation and functional coupling of these receptor isoforms.