Yanbin Liang

Prostamides (prostaglandin-ethanolamides) and their pharmacology

The prostamides are part of a large and continually expanding series of pharmacologically unique neutral lipids. They are COX‐2 derived oxidation products of the endocannabinoid/endovanniloid anandamide. Prostamide pharmacology is unique and, as in the case of the endocannabinoids anandamide and 2‐arachidonylglycerol, bears little resemblance to that of the corresponding free acids. By virtue of its close relationship to the anti‐glaucoma drug bimatoprost, prostamide F2α has received the greatest research attention. Prostamide F2α and bimatoprost effects appear independent of prostanoid FP receptor activation, according to a litany of agonist studies. Studies involving freshly isolated and separate feline iridial smooth muscle cells revealed that bimatoprost and FP receptor agonists stimulated different cells, without exception. This suggests the existence of receptors that preferentially recognize prostamide F2α. The recent discovery of prostamide antagonists has provided further support for prostamide receptors as discrete entities. The prototypical prostamide antagonists, AGN 204396 and 7, blocked the effects of prostamide F2α and bimatoprost but not those of PGF2α and FP receptor agonists in the feline iris. Second generation more potent prostamide antagonists, such as AGN 211334, should allow the role of prostamides in health and disease to be elucidated. From the therapeutics standpoint, the prostamide F2α analogue bimatoprost is the most efficacious ocular hypotensive agent currently available for the treatment of glaucoma.

Identification and pharmacological characterization of the prostaglandin FP receptor and FP receptor variant complexes.

Background and purpose: A prostamide analogue, bimatoprost, has been shown to be effective in reducing intraocular pressure, but its precise mechanism of action remains unclear. Hence, to elucidate the molecular mechanisms of this effect of bimatoprost, we focused on pharmacologically characterizing prostaglandin FP receptor (FP) and FP receptor variant (altFP) complexes.

Identification of an antagonist that selectively blocks the activity of prostamides (prostaglandin-ethanolamides) in the feline iris

The prostamides (prostaglandin‐ethanolamides) and prostaglandin (PG) glyceryl esters are biosynthesized by COX‐2 from the respective endocannabinoids anandamide and 2‐arachidonyl glycerol. Agonist studies suggest that their pharmacologies are unique and unrelated to prostanoid receptors. This concept was further investigated using antagonists.

Upregulation of orphan nuclear receptor Nur77 following PGF2α, Bimatoprost, and Butaprost treatments. Essential role of a protein kinase C pathway involved in EP2 receptor activated Nur77 gene transcription

Using gene chip technology, we first identified that PGF2α (FP agonist) and Butaprost (EP2 agonist) induced about a five‐fold upregulation of Nur77 mRNA expression in hFP‐HEK 293/EBNA and hEP2‐HEK293/EBNA cells. Northern Blot analysis revealed that PGF2α‐ and Butaprost‐induced upregulation of Nur77 expression are dose‐ and time‐dependent. Both PGF2α and Butaprost upregulated Nur77 gene expression through the protein kinase C (PKC) pathway. These data are the first showing a link between EP2 receptor stimulation and protein kinase C activation. Calcineurin was found to be involved downstream of the PKC pathway in PGF2α‐induced Nur77 expression, but not in Butaprost‐induced Nur77 expression. We also used Nur77 as a marker gene to compare the effects of PGF2α, Butaprost, and Bimatoprost (a prostamide) on Nur77 expression in human primary trabecular meshwork and ciliary smooth muscle (SM) cells, which are target cells for antiglaucoma drugs. The results showed that PGF2α and Butaprost, but not Bimatoprost, induced upregulation of Nur77 expression in human TM cells. PGF2α, but not Bimatoprost, dramatically induced upregulation of Nur77 mRNA expression in human ciliary SM cells, whereas Butaprost slightly upregulated Nur77 mRNA expression in SM cells. Nur77 promoter deletion analysis indicated that PGF2α, but not Bimatoprost, activated Nur77 promoter‐luciferase reporter in hFP‐HEK 293/EBNA cells. Butaprost was less efficacious in inducing Nur77 promoter‐luciferase reporter activity in hEP2‐HEK293/EBNA cells relative to PGF2α in the comparable assay. The data for Nur77 promoter functional analysis were matched to the Northern blot analysis. It appears that PGF2α and Butaprost activate Nur77 transcription mechanisms through the activation of FP and EP2 receptor‐coupled signaling pathways, whereas Bimatoprost stimulates neither FP nor EP2 receptors.

Identification of a novel alternative splicing variant of RGS5 mRNA in human ocular tissues

Regulator of G protein signaling (RGS) proteins act as GTPase‐activating proteins (GAPs) for Gα subunits and negatively regulate G protein‐coupled receptor signaling. Using RGS5 gene‐specific RT‐PCR, we have identified a novel alternative splicing variant of RGS5 mRNA in human ocular tissues. The alternative splicing of RGS5 mRNA occurred at position +44 (GenBank NM_003617), spliced out 174 bp (+44 to +218 bp) of the coding region, and encoded an RGS5s protein with a 108 amino acid N‐terminal deletion. This study is the first to document alternative splicing of an RGS5 gene. We therefore studied RGS5 and RGS5s mRNA distribution in human tissues. In the eye, RGS5s was found to be highly expressed in the ciliary body and trabecular meshwork. It was also expressed in the kidney, brain, spleen, skeletal muscle and small intestine, but was not detectable in the liver, lung, heart. RGS5s was not found in monkey and rat ocular tissues, indicating species specificity for the eye. Comparing the recombinant RGS5 and RGS5s expression in HEK293/EBNA cells, RGS5s was present almost exclusively in the cytosolic fraction, whereas RGS5 was present in both membrane and cytosolic fractions. The data suggest that the N‐terminal of RGS5 may be important for protein translocation to the cell membrane. Both RGS5 and RGS5s antagonized the rapid phosphorylation of p44/42 MAP kinase induced by Gαi coupled cannibinoid receptor‐1 activation. RGS5, but not RGS5s, inhibited the Ca2+ signaling initiated by activation of Gαq coupled angiotensin II receptors (AT1) and prostaglandin FP receptors. Cotransfection of RGS5s with RGS5 resulted in the blockade of RGS5 actions with respect to inhibition of the signal transduction initiated by activation of both AT1 and FP receptor, suggesting that RGS5s may contain functional domains that compete with RGS5 in the regulation of the Gαq coupled AT1 and FP receptors. The unique expression pattern, cellular localization and functions of RGS5s suggest that RGS5s may play a critical role in the regulation of intracellular signaling pathways.

Prostaglandin ep4 antagonists

Prostaglandin antagonists, with their pharmacological effects, are well-known drugs capable of treating widely diffused illnesses, including pain and inflammation disorders. In recent years, a major research focus has been devoted to the identification of agents able to selectively antagonize each receptor with which prostaglandins interact. This review attempts to give a broad overview of molecules capable of selectively blocking the prostaglandin PGE2 EP4 receptor. Further therapeutic applications and uses have also been disccussed, including the first drug candidate to have reached clinical trials within the last few years.