Adrenergic Alpha 1 and Alpha 2 Binding Sites Are Present in Bovine Retinal Blood Vessels

November 1987 Vol. 2811 Investigative Ophthalmology & Visual Science A Journal of Dosic and Clinical Research Articles Adrenergic Alpha 1 and Alpha 2 Binding Sites Are Present in Bovine Retinal Blood Vessels Beth A. Forsrer, Gabryleda Ferrari-Dileo, ond Douglas PV Anderson Bovine retinal vessels have sites that specifically bind 3H-p-aminoclonidine (3H-PAC) with an apparent dissociation constant of 0.12 nm and a capacity of binding of 0.15 pmolg. In addition, these vessels have 3H-prazosin binding sites bearing a dissociation constant of 5 nm and a binding capacity of 5 pmolg. To understand the implications for retina-optic nerve vascular physiology and pathophysiology, studies of the exact location of the binding sites, the bioavailability of the adrenergic agonists, and the physiological responses to receptor stimulation in both normal and pathological states are required. The neural elements of the retina also have binding sites for 3H-PAC with an apparent dissociation constant of 0.38 nm in larger quantities (6.7 pmolg tissue) than in the vascular elements. There are also binding sites for 3H-prazosin in a lower amount than in the vascular fraction (3 pmolg tissue) with a dissociation constant of 2.4 nm. These sites are presumably related to the use of norepinephrine and dopamine as neurotransmitters by retinal neurons. Invest Ophthalmol Vis Sci 28:1741-1746, 1987 Vessels of the retina and optic disc autoregulate in order to maintain an almost constant blood flow despite changes in intraocular pressure or arterial pressure. The main mechanism in autoregulation may be the tissue level of O 2 and CO 2.' In a tissue where circulation is efficiently controlled according to local metabolic needs, there would seem to be no physiologic need for control from remote locations through neural or humoral influence, and indeed these vessels lack autonomic innervation. However, retinal vessels physiologically respond to vasoconstrictors such as angiotensin II 2-3 and norepinephrine. 2 If vascular tone is indeed affected by these agents, their ability to dilate as an autoregulatory response may be reduced, making the tissue vulnerable if the circulation is challenged, for example by systemic hypotension or elevation of intraocular pressure. From the Bascom Palmer Eye Institute, Department of Ophthalmology, University of Miami School of Medicine, Miami, Florida. Supported in part by U.S. Public Health Research Grant R01 EY-00031 awarded by the National Eye Institute, Bethesda, Maryland, and grant 5-T35-07489 awarded by the National Institutes of Health, Bethesda, Maryland. Submitted for publication: November 5, 1986. Reprint requests: Dr. Douglas R. Anderson, Bascom Palmer Eye Institute, P.O. Box 016880, Miami, FL 33101. Because such limitation of autoregulation may be the reason that some individuals suffer glaucomatous optic nerve damage, 4 while others with intact autoregulation may not surfer damage, we are interested in understanding in detail the physiology of the retina-optic nerve vasculature. Physiological and in situ studies are complicated because of the presence of compensatory responses and the small size of the vessels in question. Therefore, before proceeding to such studies, we decided to establish the plausibility of a direct influence of various vasoactive agents by establishing the presence of specific binding sites that might be physiologic receptors. In this study we show that retinal vessels specifically bind 3H-prazosin and 3H-p-aminoclonidine. Materials and Methods We used freshly enucleated bovine eyes obtained at a local abattoir. Retinas were removed and placed in 5 mm Tris-HCl buffer enriched with 0.25 M sucrose (4 C, ph 7.4). After mincing with scissors, the retinas were homogenized with a hand-held Teflon pestle in a smooth glass tube. The homogenate was poured on a nylon sieve and washed thoroughly. The material retained on the sieve was rehomogenized, sieved and washed again with fresh buffer. The new material on 1741 Downloaded From: http:iovs.arvojournals.org on 03042018

1742 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE November 1987 Vol. 28 the sieve (mainly vessels) and the material collected from both sieving steps (retinal elements) were each centrifuged at 3,000 rpm for 10 min. For the vascular fraction, the supernatant was discarded (representing pieces of retina previously trapped in the tangled vessels), and the light pellet was resuspended in fresh buffer in a pre-weighed tube, disrupted with a Brinkmann Polytron and centrifuged at 18,000 rpm for 10 min. The supernate was discarded and the tube reweighed to determine the weight of the wet pellet (average weight of 500 mg from 30-40 cow eyes). This pellet, consisting of an enriched fraction of vessels, was used for binding experiments. The supernatant from the low-speed centrifugation of the retinal fraction was homogenized with a Polytron at high speed and centrifuged at 18,000 rpm for 10 min. The resulting pellet, containing retinal neuronal tissue, 5 was used to assess binding of the radioligand in the "non-vascular fraction" (average weight of 1100 mg from 30-40 cow eyes). All the steps were performed at 4 C. Alpha-2 Adrenergic Binding Sites We used 3H-para-aminoclonidine (3H-PAC) as a selective ligand that binds selectively to alpha-2 sites at concentrations in the nm range. 6 ' 7 The paniculate fractions from vessels and non-vascular retinal tissue were separately diluted in the assay buffer consisting of 50 ram Tris-HCl buffer, ph 7.7, to give a final concentration of 100 mg per ml. Binding assays were conducted in duplicate by incubating aliquots of tissue (8-10 mg of tissue per tube) with various concentrations (0.01-1 nm) of 3Hpara-aminoclonidine (New England Nuclear Boston, MA; 45 Cimmol), for total and for non-specific binding. Non-tritiated para-aminoclonidine (Sigma Chemical Co., St. Louis, MO) was used to define non-specific binding at a concentration of 1 tm. The incubations lasted 30 min at 25 C, and were terminated by rapid filtration through glass fiber filters. Filters and retained membranes were washed three times with 5 ml fresh ice-cold assay buffer. Radioactivity retained in the filters was counted by liquid scintillation spectroscopy. Alpha-1 Adrenergic Binding Sites Membranes were prepared as described above, and incubated in 1 ml final volume of 50 mm Tris-HCl, ph 7.7, with selected concentrations of 3H-Prazosin (New England Nuclear; 28 Cimmol) at 25 C for 30 min. Nonspecific binding was established with 1 pm prazosin (Pfizer, New York, NY) prepared in 0.1% ascorbic acid. Membranes were filtered, washed, and counted as for the alpha-2 site assay. For both radioligands, the specific binding was calculated by subtracting the counts of non-specific binding (in the presence of an excess competing ligand) from the counts obtained as total binding. All data were expressed as the arithmetic mean from six to ten experiments. Saturation radioligand binding curves were analyzed by means of Scatchard plots to determine the apparent equilibrium dissociation constant (Kd) and the amount of radioligand bound at saturation (Bmax). Scanning Electron Microscopy Retinal vessels, freshly isolated as described above, were sedimented in 5 mm Tris-HCl buffer, 0.25 M sucrose, ph 7.4. The pellets were fixed in 3% glutaraldehyde-phosphate buffer for 1 hr, and in 2% OsO 4 for an additional hour. The fixed samples were then dehydrated through several changes of increasing concentrations of ethanol and in acetone. After being dried by the critical point method, the pellets were coated with gold, and examined and photographed in a scanning electron microscope. Results The content of our retinal vascular fraction was examined by scanning electron microscope. As shown in Figure 1, the vessels appeared clean with only few small fragments of non-vascular tissue. Equilibrium Specific Binding Specific binding of 3H-PAC to the bovine vascular fraction was approximately 65% of the total binding over a range of concentrations from 0.01 to 1 nm (Fig. 2A). Scatchard analysis of the binding isotherm indicates the presence of a binding site (Fig. 2B), with an apparent dissociation constant (Kd) of 0.1 nm, and a total number of sites of 0.15 pmol per gram of wet vascular tissue isolated. In the retinal non-vascular homogenates, the specific binding of 3H-PAC represented almost 90% of the total (Fig. 3A). Scatchard analysis indicates the presence of binding sites with a Kd of 0.38 nm and a Bmax of 6.7 pmolg of wet non-vascular tissue (Fig. 3B). Specific binding of 3H-prazosin to bovine retinal vessels and retina homogenates saturated at 10-20 nm concentration of the radioligand (Figs. 4A, 5A). Scatchard analysis of the data from five individual Downloaded From: http:iovs.arvojournals.org on 03042018

No. 11 ADRENERGIC DINDING SITES IN RETINAL VESSELS Forsrer er al. 1743 Fig. I. Scanning electron micrograph of the isolated bovine retinal vessels used for the binding experiments in the "vascular fraction". Magnification XI400. experiments with retinal vessels indicates a single population of binding sites, in the range of concentrations studied, with a Kd of 5 nm and Bmax of 5 pmolg (Fig. 4B). The non-vascular retinal homogenates also have a rectilinear Scatchard plot (Fig. 5B) which shows a single binding site with a Kd of 2.4 nm and a capacity of binding of 3 pmo!g. Discussion The main finding of the present study was that both the retinal vessels and the retina itself possess specific binding sites for the alpha-1 selective agonist prazosin and for the alpha-2 selective partial agonist para-am inoclonidine. When concentrations higher Kd = 0.12 nm Fig. 2. (A) Total ( ) and specific (O) binding of 3HPAC to bovine retinal vessels homogenates as a function of ligand concentration (0.03-1 nm). Non-specific binding was defined by 1 nm PAC. (B) Scatchard plot of the specific binding. All points represent the mean of ten independent experiments carried out in duplicate. Kd = 0.12 nm; Bmax = 1.5 pm(0.15 pmolg). Bmax = 1.49 pm (0.15 pmolg) FREE H-PAC (nm) Downloaded From: http:iovs.arvojournals.org on 03042018 BOUND 3H-PAC (pm)

1744 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE November 1987 Vol. 28 UJ c O CD 0.2? B. - x * X Kd = 0.38 Bmax = 66.9 pm (6.7 pmolg) \ \ Fig. 3. (A) Total ( ) and specific binding (O) of 3H- PAC to retinal homogenates ("non-vascular fraction"). (B) Scatchard plot of the specific binding. Every point is the mean often separate experiments performed in duplicate. Kd = 0.38 nm; Bmax = 66.9 pm (6.7 pmolg). FREE H-PAC (nm) 1 1 20 BOUND J H-PAC 40 60 (pm) 3 (0 0> o> E [fmol z (> O N< a. Q. 1 X > Q O 00 14 12 10 8 6 A r <±* 4 r J x \ IS 21- \J %y i i i i _^ o o 1 5 X UJ UJ 10 cr u. Q o 5 CD B Kd = 5 nm. Bmax ' 52 pm (5 pmolg) i i i i i i i ^^w. 10 15 20 10 20 30 40 50 FREE H-PRAZOSIN (nm) BOUND 3 H-PRAZOSIN (pm) Fig. 4. (A) Total ( ) and specific (O) binding of 3Hprazosin binding to retinal vessel membrane suspensions. Non-specific binding was defined by 1 nm prazosin. Each point is the mean of five separate experiments performed in duplicate. (B) Scatchard analysis of the specific binding. Kd = 5 nm; Bmax = 5 pmolg wet tissue. o X UJ Z> O CD 15 10 5 B Kd = 2. 4 nm Bmax = 29 pm (3 pmolg) Fig. 5. (A) Total ( ) and specific binding (O) of 3Hprazosin to retinal homogenates. (B) Scatchard analysis of the specific binding. Kd = 2.4 nm; Bmax = 3 pmolg wet tissue. Values represent means of duplicate determinations from five separate experiments. \ 10 15 20 10 20 30 FREE H-PRAZOSIN (nm) BOUND 3 H-PRAZOSIN (pm) 1 Downloaded From: http:iovs.arvojournals.org on 03042018

No. 11 ADRENERGIC DINDING SITES IN RETINAL VESSELS Forsrer er ol. 1745 than 1 nm of the partial agonist para-aminoclonidine were used in our preparation, and when cations and guanine nucleotides were used to modify the binding, it appeared as if two different affinity states might be present in both the vascular and non-vascular fractions for the para-aminoclonidine binding site (unpublished data). However, we did not perform any further detailed analyses, such as kinetic studies, to prove the presence of such different conformations for the para-aminoclonidine binding site. The presence of specific adrenergic binding sites in the retinal homogenates is not an unexpected finding. This is a tissue that contains norepinephrine and the enzymatic machinery for its synthesis 8 ; in addition, alpha adrenergic receptors can be stimulated by dopamine, which is present in large amounts in the inner retina. This finding, and the value for the affinity of the binding site, agree with previous reports on the presence of alpha-2 adrenergic receptors in bovine retinas 910 and on the presence of both alpha-1 and alpha-2 adrenoreceptors in rat retinal sections." Since catecholaminergic amacrine cells are present in the retina, the alpha-2 binding sites found in the present study may be either postsynaptic or presynaptic. The alpha-1 and alpha-2 binding sites in the retinal vessels represent a new, but again not unexpected finding. Postsynaptic alpha receptors are present in both peripheral and central arteries as a mixed population of alpha-1 and alpha-2 adrenoreceptors, both subserving contractile responses. The mechanism for each receptor appears to be different. The alpha-2 type is negatively coupled to adenylate cyclase and critically dependent on extracellular Ca 2+ to elicit its vasoconstrictor response, 1213 whereas the alpha-1 type appears to depend on intracellular Ca 2+ and phosphatidyl inositol turnover. 1415 The binding sites in retinal vessels were few in number, but with high affinity and assuming they are receptors, they may be capable of producing a contractile response to low concentrations of adrenergic agonists. As there is no adrenergic innervation at these arteries, there is no potential stimulation from the autonomic nervous system. The normal amount of circulating catecholamines is in the nanomolar range, which is enough to stimulate both alpha-1 and alpha-2 receptors, and enough to maintain smooth muscle tone efficiently. However, it is not clear how the intraluminal catecholamines might reach smooth muscle alpha-adrenergic sites in retinal arterioles, or the intramural pericytes of the capillaries, because these retinal vessels lack fenestrations and possess tight junctions in the endothelium. Adrenergic agonists might reach the pericytes or the smooth muscle only where there is a breach in the blood-retinal barrier. Another potential source for the activation of muscular alpha receptors is diffusion of excess neurotransmitter from the adjacent retina. This is an unlikely normal event, because re-uptake and catabolism are efficient mechanisms for prevention of such spill-over. The alpha-adrenergic binding sites may not be located in the muscular layer but in the endothelium. Evidence for the presence of alpha-2 adrenergic receptors in the luminal layers has been presented for peripheral and central arteries. 1617 According to some of these reports, the alpha-2 receptors located in the luminal side promote a relaxing response or at least attenuate a preexisting contractile one. Therefore, to understand the implications of the present experiments, we must next identify the exact location of the binding sites and demonstrate the type of physiologic response to agonists' stimulation. In conclusion, vessels of the retina have specific sites for binding of catecholamines, angiotensin 18 and perhaps other vasoactive agents. Further studies are needed to locate these sites on specific cells, to understand if these sites are functional receptors that help maintain hemodynamic homeostasis, and to determine if their stimulation modifies the autoregulatory capabilities of the vessels under physiologic or pathologic circumstances. Key words: prazosin binding sites, para-aminoclonidine binding sites, retinal blood vessels, retina Acknowledgment Prazosin was made available through the courtesy of Pfizer Laboratories Division, New York, New York. References 1. Tsacopoulos M: Regulation of retinal blood flow. In Scientific Foundations of Ophthalmology, Perkins ES and Hill DW, editors. London, William Heinemann Medical Books Ltd., 1977, pp.44-49. 2. Dollery CT, Hill DW, and Hodge JV: The response of normal retinal blood vessels to angiotensin and noradrenaline. J Physiol 165:500, 1963. 3. Rockwood EJ, Fantes F, Davis EB, and Anderson DR: The response of retinal vasculature to angiotensin. Invest Ophthalmol Vis Sci 28:676, 1987. 4. Anderson DR: The posterior segment of glaucomatous eyes. In Basic Aspects of Glaucoma Research, Liitjen-Drecoll E, editor. Stuttgart, Schattner, 1982, pp. 167-190. 5. Redburn DA: Uptake and release of 14C-GABA from rabbit retina synaptosomes. Exp Eye Res 25:265, 1977. 6. Rouot BR and Snyder SH: 3H-para-aminoclonidine: A novel ligand which binds with high affinity to alpha-adrenergic receptors. Life Sci 25:769, 1979. Downloaded From: http:iovs.arvojournals.org on 03042018

1746 INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE November 1987 Vol. 28 7. Atlas D and Sabol SL: Interactions of clonidine and clonidine analogues with alpha-adrenergic receptors of neuroblastoma X glioma hybrid cells and rat brain. Eur J Biochem 113:521, 1981. 8. Cohen J and Hadjiconstantinou M: Identification of epinephrine and phenylethanolamine-n-methyl transferase in rat retina. Fed Proc 433:2725, 1984. 9. Bittinger H, Heid J, and Wigger N: Are only alpha 2 adrenergic receptors present in bovine retina? Nature 287:645, 1980. 10. Osborne NN: Binding of 3H-noradrenaline to bovine membranes of the retina: Evidence of the existence of alpha-2 receptors. Vision Res 22:1401, 1982. 11. Zarbin MA, Wamsley JK, Palacios JM, and Kuhar MJ: Autoradiographic localization of high affinity GABA, benzodiazepines, dopamine, adrenergic and muscarinic cholinergic receptors in rat, monkey and human retina. Brain Res 374:75, 1986. 12. Mathews W, Jim K, Hieble P, and DeMarinis R: Postsynaptic alpha-adrenoreceptors on vascular smooth muscle. Fed Proc 43:2923, 1984. 13. Ruffolo RR Jr: Interactions of agonists with peripheral alphaadrenergic receptors. Fed Proc 43:2910, 1984. 14. Fain JN and Garcia-Sainz JA: Role of phosphatidylinositol turnover in alpha-1 and of adenylate cyclase inhibition in alpha-2 effects of catecholamines. Life Sci 26:1183, 1980. 15. Zeleznikar RJ Jr, Quist EE, and Drewes LR: An alpha-1 adrenergic receptor-mediated phosphatidylinositol effect in canine cerebral microvessels. Mol Pharmacol 24:163, 1983. 16. Cocks M and Angus J: Endothelium-dependent relaxation of coronary arteries by noradrenaline and serotonin. Nature 305:627, 1983. 17. Egleme C, Godfraind T, and Miller RC: Enhanced responsiveness of rat isolated aorta to clonidine after removal of the endothelial cells. Br J Pharmacol 81:16, 1984. 18. Ferrari-Dileo G, Davis EB, and Anderson DR: Angiotensin binding sites in bovine and human retinal blood vessels. Invest Ophthalmol Vis Sci 28:1747, 1987. Downloaded From: http:iovs.arvojournals.org on 03042018