Parimal Bhattacherjee

Quantification of ocular inflammation: Evaluation of polymorphonuclear leucocyte infiltration by measuring myeloperoxidase activity

Myeloperoxidase (MPO) activity was measured in rabbit cornea and iris-ciliary body to quantitate the infiltration and accumulation of polymorphonuclear leucocytes (PMNs) following an inflammatory stimulus. Following injection of clove oil into the cornea, MPO activity could be detected in the cornea at 6 hr, reaching a maximum at 12 hr, and falling to non-detectable levels at 72 hr. MPO activity was only detected in the iris-ciliary body 24 hr after intracorneal clove oil injection. MPO activity in the iris-ciliary body increased in a dose-dependent manner following intravitreal injection of endotoxin. No MPO activity could be detected in cornea. Topical administration of dexamethasone inhibited MPO activity in cornea and iris-ciliary body 24 hr after intracorneal clove oil and intravitreal endotoxin injection, respectively. Measurement of MPO activity in ocular tissues could provide a useful tool to quantitatively evaluate the severity and time course of inflammation.

Detection of EP2, EP4, and FP receptors in human ciliary epithelial and ciliary muscle cells

We have examined the expression of three prostaglandin (PG) receptors, EP2, EP4, and FP, in a nonpigmented ciliary epithelial cell line (ODMCl-2) and in human ciliary muscle (HCM) cells. Total RNA preparations from either ODMCl-2 or HCM cells were subjected to reverse transcription-polymerase chain reaction (RT-PCR) with sense and antisense primers for each of the three PG receptors. The RT-PCR generated DNA products of predicted sizes corresponding to the EP2, EP4, and FP receptors in both ODMCl-2 and HCM cells. PCR products corresponding to each receptor were hybridized with specific 32P-labeled probes and, for further confirmation, digested with appropriate restriction enzymes. Pharmacological studies with the EP2 receptor-selective agonist butaprost resulted in a significant increase in the cyclic AMP level in ODMCl-2 cells. The stimulation of cyclic AMP in ODMCl-2 cells by PGE2 and 11-deoxy PGE1, the respective EP1/EP2/EP3/EP4 and EP2/EP3/EP4 receptor agonists, was concentration-dependently inhibited by the EP4 receptor-selective antagonist AH23848. These results conclusively demonstrate the presence of both mRNA and protein for EP2, EP4, and FP receptors in ODMCl-2 and HCM cells.

Biosynthesis of lipoxygenase products by ocular tissues

The metabolism of arachidonic acid via the lipoxygenase pathway has been investigated in conjunctival and iris tissue taken from eyes of various species. In addition we have also studied two inhibitors of arachidonate metabolism, BW 755 and indomethacin, on albino rabbit ocular tissues. The ocular tissues of most species (monkey, dog, cat, rabbit, guinea-pig and rat) formed lipoxygenase products from exogenous arachidonic acid. The exception was the albino rabbit iris, where no lipoxygenase product was detected. The major lipoxygenase product found was 12-HETE, although 5-HETE and 5,12-diHETE were formed to a lesser extent by the conjunctiva and iris of the Dutch rabbit. The rat ocular tissues and guinea pig conjunctiva also formed 5-HETE. In the conjunctiva of the albino rabbit, indomethacin was a relatively specific inhibitor of the cyclo-oxygenase pathway whereas BW 755 inhibited both the cyclo-oxygenase and lipoxygenase pathways of arachidonic acid metabolism. In view of the possible roles of lipoxygenase products in inflammatory reactions and the ability of ocular tissues to synthesize these products, dual inhibitors of cyclo-oxygenase and lipoxygenase pathways may be useful agents to control ocular inflammatory responses.

Anti-inflammatory effects of betamethasone phosphate, dexamethasone phosphate and indomethacin on rabbit ocular inflammation induced by bovine serum albumin

Following the induction of immunogenic uveitis betamethasone and dexamethasone reduced conjunctiva and iris hyperemia and aqueous flare. Indomethacin reduced iris hyperemia, but potentiated conjunctival hyperemia. All three agents inhibited the increase in the protein content of the aqueous that follows induction of immunogenic uveitis. Betamethasone was slightly more potent than dexamethasone and indomethacin. White cell entry into the aqueous was inhibited by betamethasone and dexamethasone. Paradoxically, however, indomethacin significantly potentiated the number of polymorphonuclear leukocytes (PMNs) entering the aqueous. The anti-inflammatory effects of steroidal agents may be due to the inhibition of the release of arachidonic acid (AA) which is a precursor of both lipoxygenase and cyclooxygenase products. The potentiation of the PMN response by indomethacin in immunogenic uveitis may be due to an inhibition of cyclooxygenase product formation and a facilitation of lipoxygenase products (which are potent chemotactic agents) from AA.

Generation second messengers by prostanoids in the iris-sphincter and ciliary muscles of cows, cats and humans

We have examined the generation of second messengers after stimulation of feline, bovine, human iris-sphincter and ciliary muscles by selected prostaglandins (PGs). The tissues, labeled or unlabeled with 3H-myo-inositol, were stimulated by a range of concentrations of 16,16-dimethyl PGE2, 11-deoxy PGE1, 17-phenyl trinor PGE2 and PGF2alpha. In both tissues of all three species, 16,16-dimethyl PGE2 and 11-deoxy PGE1 stimulated the formation of cyclic AMP. Butaprost, an EP2 receptor agonist, which was tested only in feline ciliary muscle, generated cyclic AMP. In the feline iris-sphincter and in bovine and feline ciliary muscles, 17-phenyl trinor PGE2, an EP1 receptor agonist, significantly increased inositol phosphate turnover. The FP receptor agonist, PGF2alpha stimulated inositol phosphate turnover in the bovine, feline, and human iris-sphincter muscles and in human ciliary muscles. Feline and bovine ciliary muscles did not respond to PGF2alpha. These results suggest that EP1 receptors are present in feline iris-sphincter muscle and in bovine and feline ciliary muscles. The EP2 receptors exist in both tissue. These results also suggest the presence FP receptors in bovine, feline, and human iris-sphincter and in human ciliary muscles. Bovine and feline ciliary muscles do not appear to express FP receptors.

Prostaglandin E2 binding site distribution and subtype classification in the rabbit iris-ciliary body

The distribution and characteristics of specific binding sites for tritium labeled prostaglandin E2 (3H-PGE2) were examined in membrane preparations from rabbit iris-sphincter, iris and ciliary body. The majority of 3H-PGE2 specific binding sites were found in the ciliary body (46%) followed by the iris (37%) and the iris-sphincter muscle (5%). Scatchard analysis of saturable 3H-PGE2 binding sites in the ciliary body indicated a single binding site with a Kd of 2.81 nM and Bmax value of 84 fmoles bound/mg protein. Competition by agonists selective for the EP1, EP2 and EP3 receptor subtypes of the EP (PGE2) prostanoid receptor indicated that the majority of rabbit ciliary body 3H-PGE2 binding sites are of the EP2 subtype. Incomplete displacement of labeled 3H-PGE2 from its binding sites by the EP2 selective agonist 11-deoxy PGE1 suggests the presence of additional EP or non-EP binding sites. There was essentially no binding to EP1 receptor sites as defined by the displacement of 3H-PGE2 by 17-phenyl-trinor PGE2. A weak displacement of 3H-PGE2 by the EP3/EP1 specific agonist, sulprostone, may account for the presence of a small number of EP3 specific binding sites in this tissue. The predominant distribution of PGE2 binding sites in the ciliary body and their identification as EP2 selective, supports recent functional studies where topical application of prostanoids with EP2 but not EP1 or EP3 agonist activity resulted in breakdown of the blood-aqueous barrier.

Studies on receptor binding and signal transduction pathways of unoprostone isopropyl

We examined the binding characteristics of unoprostone isopropyl and its metabolite, M1 (M1), in bovine corpus luteum membranes, mobilization of intracellular calcium in human ciliary muscle cells and cyclic AMP generation in rabbit iris-ciliary body. The ligand binding assay of 3H-unoprostone isopropyl and M1 did not demonstrate any specific binding of these compounds in the bovine corpus luteum membranes. However, there was a high specific binding of prostaglandin F2alpha. Competitive ligand binding studies showed that neither the docosanoid, unoprostone isopropyl, nor M1 binds to prostaglandin receptor sites. In human ciliary muscle cells that express EP1, EP2 and FP receptors, unoprostone isopropyl did not increase the mobilization of intracellular calcium nor was it able to generate cyclic AMP at low concentrations in rabbit iris-ciliary body. Similar observations were made with M1 on the above signal transduction pathways. From these results, it is concluded that unoprostone isopropyl and M1 do not bind to prostaglandin (PG) receptor sites in the bovine corpus luteum membranes and do not have affinity for PG receptors linked to intracellular calcium and cyclic AMP second messenger systems.

Comparative studies on prostanoid receptors in human non-pigmented ciliary epithelial and mouse fibroblast cell lines

To examine the expression of functional prostanoid receptors in the human non-pigmented ciliary epithelial (ODMC1-2) and mouse fibroblast cell lines (NIH 3T3) we have measured the generation of the second messengers, cyclic AMP, inositol phosphates and the mobilization of intracellular calcium ([Ca2+]i) following stimulation by prostaglandin receptor agonists. The amount of cyclic AMP generated was measured by a protein binding method. Radiolabeled inositol phosphates were separated using ion exchange columns and quantified by counting the radioactivity. For intracellular calcium measurements, Fura 2-AM loaded cells were stimulated by PG receptor agonists and the calcium activated fluorescence was measured in a spectrofluorometer. In the ODMC1-2 cell line, the formation of cyclic AMP was stimulated by prostaglandin E2, butaprost and 11-deoxy-prostaglandin E1. The stimulation of cyclic AMP production by prostaglandin E2 was partially inhibited by the EP4 receptor antagonist AH23848. Prostaglandin E2 and 11-deoxy-prostaglandin E1 stimulated the formation of cyclic AMP in NIH 3T3 cells. In ODMC1-2 cells, total inositol phosphate turnover was not increased by 17-phenyl-trinor-prostaglandin F2 alpha, 17-phenyl-trinor-prostaglandin E2 or 11-deoxy-prostaglandin E1. In contrast, all these agonists, with the exception of 11-deoxy-prostaglandin E1, significantly increased total inositol phosphates in NIH 3T3 cells. In the NIH 3T3 cell line, only prostaglandin F2 alpha and 17-phenyl-trinor-prostaglandin F2 alpha increased [Ca2+]i in a dose-dependent manner; in ODMC1-2 cells, neither these agonists nor 17-phenyl-trinor-prostaglandin E2 increased [Ca2+]i. The present studies suggest that in ODMC1-2 cells, EP2 and EP4 receptors but neither EP1 nor FP receptors are expressed; these studies also imply, NIH 3T3 cells express EP2 and FP receptors, while EP1 receptors appear to be absent in this cell line.

Blood-aqueous barrier in prostaglandin EP2 receptor knockout mice

The role of prostaglandin EP 2 receptors in the disruption of the blood-aqueous barrier was examined using EP 2 receptor-deficient mice. Eyes were topically treated with EP receptor agonists or subjected to paracentesis. Fluorescein angiography was performed after topical treatment with 2.0µg butaprost. The results show that EP receptor agonists, PGE 2 and the EP 2 receptor-selective agonist butaprost, increased aqueous humor protein in EP 2 +/+ wild-type mice to 18.0mg/ml and 12.0 mg/ml, respectively, from the control value of 2.7 mg/ml. The increase in aqueous humor protein concentration in response to these EP receptor agonists was reduced significantly in EP 2 receptor-deficient mice. Fluorescein leakage into the anterior chamber, two minutes after its injection, was significantly greater in butaprost-treated wild-type mice than in butaprost-treated knockout mice. Protein concentration, 15 min after paracentesis, increased from 2.2mg/ml to 25.0 mg/ml in the aqueous humor of the eyes of wild-type mice, while the increase in knockout mice was 10.6 mg/ml. These results suggest that EP 2 and EP 4 receptors mediate the disruption of the blood-aqueous barrier induced by EP receptor agonists and paracentesis.

Effects of ebselen on arachidonate metabolism by ocular and non-ocular tissues

The formation of cyclooxygenase products in rabbit and rat ocular and non-ocular tissues in vitro, detected by radio-thin-layer chromatography, was inhibited in a concentration-dependent manner by ebselen (PZ 51), an anti-inflammatory seleno-organic compound which has glutathione peroxidase and anti-oxidant activities. The exception was prostaglandin F2 alpha (PGF2 alpha) formation in the rabbit irisciliary body which was stimulated by ebselen in the concentration range 2-10 microM. These observations were confirmed by gas chromatography-mass spectrometry. The concentration that inhibited 50% of prostaglandin biosynthesis (IC50) in the rabbit iris-ciliary body was 9.3 microM. Ebselen also inhibited the formation of 12-hydroxyeicosatetraenoic acid (12-HETE) in rabbit and rat ocular tissues and rabbit platelets. The IC50 in the rabbit cornea was 4 microM, whereas higher concentrations were generally required to achieve similar inhibition in other tissues. The formation of 12-HETE by rabbit spleen, however, was not decreased by ebselen at concentrations that were inhibitory in other tissues.