JOURNAL OF OCULAR PHARMACOLOGY Volume 7, Number 2, 1991 Mary Ann Liebert, Inc., Publishers
Arachidonic Acid Metabolism in Human and Bovine Retina PRASAD S. KULKARNI Department of Ophthalmology and Visual Science, Kentucky Lions Eye Research Institute, University of Louisville School of Medicine, Louisville, Kentucky
ABSTRACT
Arachidonic acid (AA) metabolism in the human and bovine retina was assessed. demonstrates that the human and bovine retina can synthesize prostaglanThis tissue also F2a, I2, D2 and thromboxane A2 from UC-AA. synthesized leukotriene (LT) B,, and 12-hydroxyeicosatetraenoic acid (12-HETE) Although human and bovine retina metabolized AA into PGs and LTs, the ability to synthesize these products was considerably less than some other ocular tissues of various species. This din
study
.
INTRODUCTION
The membrane phospholipids of mammalian cells are enriched with arachidonic a C20:4 polyunsaturated fatty acid (1). Following stimulation of phospholipase A2, AA is released and metabolized into the cyclooxygenase products: acid
(AA),
prostaglandin (PG)E2, PGF2a, PGI2, thromboxane (Tx)A2, (1) and is also metabolized into the 5 and 12 lipoxygenase products: leukotriene (LT) B,,, LTC,,, LTD4, LTEA, LTF4 and 12-hydroxyeicosatetraenoic acid (12-HETE) (2). Some PGs and LTs have important biological activities and are implicated in the regulation of cardiovascular dynamics, renal excretion, neuromodulation, and ocular inflammation (1,2). However, the biological activities of these products relevant to retinal function are not yet established. AA metabolism into cyclooxygenase and lipoxygenase products in different ocular tissues of various species has been demonstrated (3). However, only a few studies have demonstrated the presence of cyclooxygenase and lipoxygenase pathways in rabbit (4), bovine and dog retina (5). These tissues synthesized very small In the present study, we amounts of eicosanoids from exogenously added AA. determined the ability of the human retina to synthesize eicosanoids and compared this synthesis to that of the bovine retina. MATERIALS AND METHODS
Fresh normal human retinas were supplied by the Kentucky Lions Eye Bank, at Louisville. Human eyes were obtained about 2-4 hr postmortem. the removal of the cornea (for keratoplasty) the retina was excised and frozen at -70°C (< to 15 days). Previously we have shown that anterior uvea of human eyes enucleated within 6 hr postmortem synthesize substantial amounts
University of Immediately after the
135
of cyclooxygenase and lipoxygenase products (6). In each sample, 3-4 retina (300 Fresh 500 mg wet wt) were pooled and used to determine enzymatic activities. bovine eyes were obtained from a local slaughterhouse and brought to the laboratory on ice. Retinas were isolated, weighed and cut into small pieces. Amounts (300 500 mg wet wt) of bovine retina used were similar to human retina. The procedure to determine the synthesis of cyclooxygenase and lipoxygenase products from 14C-AA has been described previously (6). In our earliest studies a 30-min on the synthesis of eicosanoids from UC-AA in human anterior uvea, incubation period was used, and these studies showed that this tissue did not In a later study, synthesize detectable amounts of radiolabelled products. however, when the uveal tissue was incubated for a longer time (2 hrs), detectable amounts of all the products were synthesized (6). Therefore in the current study, both human and bovine tissues were incubated with 1AC-AA (500 nCi, specific activity 40 mM/mCi, Amersham) in oxygenated Krebs-Henseleit solution at 37°C for 2 hrs. Enzymatic activities in retinal tissues were stimulated by addition of Ca"1"* ionophore A23187 (20 ¿ig/ml) and epinephrine (500 /jg/ml) to the incubation medium (6). At the end of the incubation period, the reaction was stopped by adding 10% formic acid. Extraction and separation of metabolites by thin layer chromatography (TLC) has been described previously (6). The developed chromatogram was exposed to x-ray film (Kodak XR-7) for ten days. The film was then developed, and with the TLC plate and autoradiogram accurately superimposed, areas of AA-products were marked and scraped off the plate for quantitative measurements of uC-content by liquid scintillation counting. In another series of experiments, we scraped off silica gel corresponding to the spots from 5, 12-diHETE and PGE2 on the TLC plate. These products were then eluted into chloroform: methanol (2:1) solvent. The organic solvent was evaporated and the residue was dissolved in saline and the concentrations of PGE2 and LTB
Arachidonic Acid Metabolism in Human and Bovine Retina PRASAD S. KULKARNI Department of Ophthalmology and Visual Science, Kentucky Lions Eye Research Institute, University of Louisville School of Medicine, Louisville, Kentucky
ABSTRACT
Arachidonic acid (AA) metabolism in the human and bovine retina was assessed. demonstrates that the human and bovine retina can synthesize prostaglanThis tissue also F2a, I2, D2 and thromboxane A2 from UC-AA. synthesized leukotriene (LT) B,, and 12-hydroxyeicosatetraenoic acid (12-HETE) Although human and bovine retina metabolized AA into PGs and LTs, the ability to synthesize these products was considerably less than some other ocular tissues of various species. This din
study
.
INTRODUCTION
The membrane phospholipids of mammalian cells are enriched with arachidonic a C20:4 polyunsaturated fatty acid (1). Following stimulation of phospholipase A2, AA is released and metabolized into the cyclooxygenase products: acid
(AA),
prostaglandin (PG)E2, PGF2a, PGI2, thromboxane (Tx)A2, (1) and is also metabolized into the 5 and 12 lipoxygenase products: leukotriene (LT) B,,, LTC,,, LTD4, LTEA, LTF4 and 12-hydroxyeicosatetraenoic acid (12-HETE) (2). Some PGs and LTs have important biological activities and are implicated in the regulation of cardiovascular dynamics, renal excretion, neuromodulation, and ocular inflammation (1,2). However, the biological activities of these products relevant to retinal function are not yet established. AA metabolism into cyclooxygenase and lipoxygenase products in different ocular tissues of various species has been demonstrated (3). However, only a few studies have demonstrated the presence of cyclooxygenase and lipoxygenase pathways in rabbit (4), bovine and dog retina (5). These tissues synthesized very small In the present study, we amounts of eicosanoids from exogenously added AA. determined the ability of the human retina to synthesize eicosanoids and compared this synthesis to that of the bovine retina. MATERIALS AND METHODS
Fresh normal human retinas were supplied by the Kentucky Lions Eye Bank, at Louisville. Human eyes were obtained about 2-4 hr postmortem. the removal of the cornea (for keratoplasty) the retina was excised and frozen at -70°C (< to 15 days). Previously we have shown that anterior uvea of human eyes enucleated within 6 hr postmortem synthesize substantial amounts
University of Immediately after the
135
of cyclooxygenase and lipoxygenase products (6). In each sample, 3-4 retina (300 Fresh 500 mg wet wt) were pooled and used to determine enzymatic activities. bovine eyes were obtained from a local slaughterhouse and brought to the laboratory on ice. Retinas were isolated, weighed and cut into small pieces. Amounts (300 500 mg wet wt) of bovine retina used were similar to human retina. The procedure to determine the synthesis of cyclooxygenase and lipoxygenase products from 14C-AA has been described previously (6). In our earliest studies a 30-min on the synthesis of eicosanoids from UC-AA in human anterior uvea, incubation period was used, and these studies showed that this tissue did not In a later study, synthesize detectable amounts of radiolabelled products. however, when the uveal tissue was incubated for a longer time (2 hrs), detectable amounts of all the products were synthesized (6). Therefore in the current study, both human and bovine tissues were incubated with 1AC-AA (500 nCi, specific activity 40 mM/mCi, Amersham) in oxygenated Krebs-Henseleit solution at 37°C for 2 hrs. Enzymatic activities in retinal tissues were stimulated by addition of Ca"1"* ionophore A23187 (20 ¿ig/ml) and epinephrine (500 /jg/ml) to the incubation medium (6). At the end of the incubation period, the reaction was stopped by adding 10% formic acid. Extraction and separation of metabolites by thin layer chromatography (TLC) has been described previously (6). The developed chromatogram was exposed to x-ray film (Kodak XR-7) for ten days. The film was then developed, and with the TLC plate and autoradiogram accurately superimposed, areas of AA-products were marked and scraped off the plate for quantitative measurements of uC-content by liquid scintillation counting. In another series of experiments, we scraped off silica gel corresponding to the spots from 5, 12-diHETE and PGE2 on the TLC plate. These products were then eluted into chloroform: methanol (2:1) solvent. The organic solvent was evaporated and the residue was dissolved in saline and the concentrations of PGE2 and LTB
