Prasad S. Kulkarni

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.

The synthesis of cyclooxygenase products in ocular tissues of various species

In the present study we compared the synthesis of various cyclooxygenase products in albino rabbit, dog (puppy and adult), cat, cow and human ocular tissues from 14C-arachidonic acid (AA). Albino rabbit conjunctiva synthesized a substantial amount (47%) of cyclooxygenase products, while cat and dog (puppy as well as adult) conjunctival tissues synthesized lesser but significant amounts of cyclooxygenase products from 14C-AA. Cyclooxygenase activity in the anterior uvea of different species tested was in the following order of potency: cat greater than albino rabbit greater than dog greater than human greater than cow. Furthermore, human and cow anterior uvea synthesized significantly lesser amount of cyclooxygenase products from 14C-AA than amounts from an intermediate substrate, 14C-PGH2. Interestingly, cow anterior uvea synthesized PGF2 alpha and 6-keto-PGF1 alpha (a stable metabolite of PGI2) while human anterior uvea synthesized all cyclooxygenase products from 14-C-PGH2.

Eicosapentaenoic acid metabolism in human and rabbit anterior uvea

Eicosapentaenoic acid (EPA) metabolism into 3 series cyclooxygenase and 5 series lipoxygenase products was assessed in human and rabbit anterior uvea. Both tissues synthesized 3 series cyclooxygenase products such as delta17 6-keto-PGF1 (PGI3 metabolite), PGE3 alpha, PGE3, PGD3 and TxB3 (a stable product of TxA3) and lipoxygenase products 12-hydroxyeicosapentaenoic acid (HEPE), 5-HEPE and 5,12-diHEPE from 14C-EPA. EPA-derived cyclooxygenase product synthesis was considerably greater than the formation of lipoxygenase products from EPA in both tissues.

Effects of prostaglandins and thromboxane A2 on the coronary circulation of adult dogs and puppies

Intracoronary injections of prostaglandin endoperoxide (PGH2), prostacyclin (PGI2) and thromboxane A2 (TXA2) were given to adult dogs and puppies three months or younger. Both PGH2 and PGI2 caused dose-related coronary vasodilation in the adult as well as in the puppy dogs; and PGH2 was about twice as potent as PGI2. The threshold dose of PGH2 for coronary vasodilation, 0.01-0.02 microgram, was the same for the adult and puppy dogs although control coronary blood flow of the adult dog was 3-10 times higher. In the pupply, maximal coronary vasodilation was effected with high doses (1.0 microgram or more) of PGI2, but not with PGH2. TXA2 prepared from PGH2 up to 1.0 microgram, had no effects on the coronary circulation of the adult dog. In contrast, both the vasocon-stricting and platelet aggregating actions of TXA2 were demonstrated in the puppy. Mechanisms for the observed age-dependent differences of the canine coronary circulation to PGH2 quantitatively, and to TXA2 qualitatively, remain to be determined.

Synthesis of cyclooxygenase products by human anterior uvea from cyclic prostaglandin endoperoxide (PGH2).

The ability of human anterior uvea to synthesize cyclooxygenase products like thromboxane-A2 (TxA2), prostaglandin (PG) I2, E2 and D2 was assessed by using radiometric chromatography and bioassay techniques. Incubation of cyclic PC endoperoxides with indomethacin-treated human anterior uvea (IHIM) produced TxA2-like activity. IHIM, when either pretreated with imidazole, a TxA2 synthetase inhibitor, or boiled, failed to generate TxA2-like activity, indicating an enzymatic nature of the endoperoxide transformation. In addition, bioassay with rabbit aorta and dog coronary artery demonstrated that human anterior uvea not only synthesizes TxA2-like but also PGI2-like activities. Other evidence that human anterior uvea is capable of generating PGI2 came from studies on the extraction and subsequent stability of the substance formed from PGH2 following incubation with IHIM. Radiometric chromatography further confirmed the generation of 6-keto-PGF1α (PGI2 end product) which was inhibited by tranylcypromine (a PGI2 synthetase inhibitor). Human anterior uvea also synthetized PGF2α, PGE2 and PGD2 from 14C-PGH2. Boiled anterior uvea failed to generate these cyclooxygenase products from [14C]PGH2 indicating that the synthesis of TxA2, PGI2, E2, F2α and D2 was enzynatic in nature.

Microsomal preparations of normal bovine iris-ciliary body generate prostacyclin-like but not thromboxane-A2-like activity

Indomethacin-treated bovine iris-ciliary body microsomes (IBIM) have been studied for their ability to convert PG endoperoxides into either thromboxane-A2 (TxA2)-like or prostacyclin (PGI2)-like activity. The biological activity of the ocular tissue microsomes were compared with either indomethacin-treated human platelet microsomes (for TxA2-like activity) or rabbit aorta microsomes (for PGI2-like activity) under appropriate incubation conditions. No evidence could be found for the formation of TxA2-like activity from PG endoperoxides by the IBIM. In contrast, when the IBIM were incubated with PGH2 for 1 min at 22 degrees C without cofactors, PGI2-like activity was produced, causing profound relaxation of the isolated dog coronary artery preparation without contracting the rabbit aorta and inhibiting arachidonic acid-induced platelet aggregation. Equivalent quantities of boiled IBIM failed to alter the biological activity of PGH2 under identical conditions. Tranylcypromine (500 microgram/ml) completely abolished the appearance of PGI2-like activity. Furthermore, the PGI2-like activity found was stable for 10 min at 22 degrees C at pH 8.5 but completely lost under similar conditions at pH 5.5. It is concluded that microsomal preparations of normal bovine iris-ciliary body can synthesize PGI2-like activity in substantial amounts but not TxA2-like activity.

Anti-inflammatory effects of ketoprofen in rabbit corneal epithelial wound model

Ocular anti-inflammatory effects of ketoprofen were assessed and compared with indomethacin in the rabbit corneal epithelial wound model. Intraperitoneally (5 mg kg-1 and 50 mg kg-1) and topically (0.01%) administered ketoprofen and topical indomethacin (0.5%) prevented the release of polymorphonuclear leukocytes (PMNs) into the tear fluid of rabbits following partial de-epithelialization. However, 0.001% ketoprofen applied topically potentiated the PMN response. Topical ketoprofen at all doses tested equally inhibited prostaglandin (PG) synthesis in both conjunctiva and iris-ciliary body. In contrast, indomethacin was more effective in inhibiting PG synthesis in conjunctiva than in the iris-ciliary body. In addition, anti-inflammatory doses of ketoprofen did not interfere with wound healing after complete corneal de-epithelialization in rabbits.

Cyclo-oxygenase and lipoxygenase pathways in cynomolgus and rhesus monkey conjunctiva, anterior uvea and eyelids

The capacity of cynomolgus and rhesus monkey conjunctiva, anterior uvea and eyelids to synthesize cyclo-oxygenase and lipoxygenase products from arachidonic acid was assessed. Both conjunctiva and eyelid tissues of cynomolgus and rhesus monkeys synthesized detectable amounts of cyclo-oxygenase and lipoxygenase products, indicating the presence of cyclo-oxygenase and 5- and 12-lipoxygenase activities in these tissues. In comparison, cynomolgus anterior uvea synthesized a considerably lesser amount of cyclo-oxygenase products. The cyclo-oxygenase activity was considerably greater than the lipoxygenase activity in cynomolgus and rhesus conjunctiva and eyelids.