Expression of distinct α 1 -adrenoceptor phenotypes in the iris of pigmented and albino rabbits (original) (raw)
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Identification of beta-adrenergic receptors in the pigmented mammalian iris-ciliary body diaphragm
Experimental Eye Research, 1982
Beta-adrenergic receptors have been recently identified in the animal iris-ciliary body diaphragm (Neufield and Page, 1977) and ciliary processes (Bromberg, Gregory and Sears, 1980). The presence of melanin in non-albino animal ciliary bodies has prevented us from applying these ligand binding techniques to the study of betareceptors and their potential role in intra-ocular pressure control, despite a recent publication to the contrary (Bhargava, Makman and Katzman, 1980). As a result we have developed a simple technique to identify these receptors in the pigmented iris-ciliary body diaphragm. Iris ciliary bodies are isolated using an operating microscope and beta-receptors in the melanin-free membranes are estimated using 1251-iodohydroxybenzylpindolol(1251-HYP) in a ligand binding assay (modified from Insel and Stoolman, 1978). Eyes from freshly slaughtered rams were immediately enucleated and placed on ice. The corneas were removed with a 6-8 mm rim of sclera under the operating microscope. The iris-ciliary body diaphragm was then dissected off the underlying vitreous under direct vision. Within 1 hr of death the iris-ciliary body diaphragm was freed and placed into cold Hartman's solution at 4%. Tissue from each eye was homogenized separately in 3 ml of a buffer solution consisting of 50 mM-Tris (pH 7.5) and 1 mM-ascorbic acid (Buffer A). Tissue was homogenized initially with an Ultraturrax (75 y0 full speed, 5 x 10 set, 4%) and then 20 strokes in a ground glass hand homogenizer. One ampoule of Hyalase (1500 i.u.) was added to the pooled homogenate, mixed and allowed to stand for 1 min. The mixture was then centrifuged (3OOOg, 20 min, 4%). The pellet was resuspended in 3 ml of buffer A and further homogenized with the Ultraturrax (25% full speed, 2 x 10 set, 4%). The homogenate was loaded onto a discontinuous sucrose gradient of 5 y0 : 75 y0 and centrifuged (131000 g, 18 hr, 4%) in a Beckman SW27 rotor. This process successfully removed all melanin to the bottom of the tube and the left membrane particles at the interface. Electron microscopy has confirmed the presence of melanin-free membrane at the 5 ?, : 75'?,, interface (Fig. 1). The interface was collected, diluted in buffer A and then re-centrifuged (131000 g, 3 hr, 4'C). The pellet was then resuspended by gentle hand homogenizat'ion in a buffer containing 20 miw-Hepes (pH 7.6), Earles Balanced Salt Solution (Flow Laboratories) and @3 mM-catechol (BDH) (Assay Buffer). All procedures were performed at 4%. To detect the presence of beta-adrenergic receptors on the melanin-free membranes the ligand 125X-HYP, which has a high affinity for these receptors (Brown, Aurbach. Hauser and Trexler, 1976) was used. In these preliminary studies two fundamental properties of such receptors, namely saturability and stereospecificit'y. were used t,o study how the ligand binds to our membrane preparations. When studying the saturability of receptors it is essential to consider only t'he specifically bound 125I-HYP since this ligand also binds non-specifically to the cell membranes. To do this, total binding is estimated for a range of 1251.HYP concentrations (see Fig. 2) and then repeated in the presence of 3 x 10e6 M-propranolol.
Release and distribution of [3H]norepinephrine in nonpigmented and pigmented rabbit iris
Proceedings of the National Academy of Sciences, 1979
Unequal accumulation of drugs in pigmented and nonpigmented iris has been known for some time. However, at low concentrations of the sympathetic neurotransmitter norepinephrine, the accumulation is equal in both types of irides. The spontaneous and potassium-induced release of 3H was studied in isolated rabbit irides labeled with 0.1 micron [3H]norepinephrine ([3H]NE). The spontaneous outflow and the tissue 3H content were similar in both types of irides, but the potassium-induced release was significantly higher in nonpigmented iris. When irides were labeled with [3H]NE in the presence of 1 micron desmethylimipramine, the potassium-induced release was still significantly higher in nonpigmented than in pigmented iris, although the tissue 3H content was higher in the latter. Subcellular distribution of [3H]NE in both types of irides is presented. The data indicate that [3H]NE is distributed unequally in both types of irides in spite of equal tissue accumulation. The binding of NE by ...
Characterization of α 1Adrenoceptor Subtypes in the Eye
Experimental Eye Research, 2000
The presence of the α " -adrenoceptor subtypes in various parts of the pig and rabbit eyes was investigated using [$H]-prazosin radioligand binding. The characterization of the subtypes was achieved by performing competition experiments with various subtype selective drugs. In the pig retina, both α "A -and α "B adrenoceptors were detected and the proportion of sites was 70 % α "A -and 30 % α " Β -adrenoceptors, respectively. In the pig iris, ciliary body and choroid, which are melanin-rich tissues, the non-specific binding of [$H]-prazosin was too high to detect any of the α " -adrenoceptor subtypes. However, in the albino rabbit iris, ciliary body and retina both α "A -and α "B -adrenoceptors were detected. The proportion of sites in the iris was 60 % α "A -and 40 % α "B -adrenoceptors, respectively. In the ciliary body and rabbit retina the proportion of sites were 70 % α "A -and 30 % α " Β -adrenoceptors. Only the α " Α -adrenoceptor subtype was detected in the rabbit choroid.
Human retinal pigment epithelial cells possess β2-adrenergic receptors
Experimental Eye Research, 1992
6adrenergic receptors on cultured human retinal pigment epithelium were demonstrated by the binding of ['"51]cyanopindolol. Its pharmacologic specificity was also examined. Specific [1251]cyanopindolol binding was saturable, with a dissociation constant of 130 PM and a receptor density of 12 fmol per onehalf million cells, which is equivalent to 14 000 receptor sites per retinal pigment epithelial cell. Agonists competed for specific [1251]cyanopindolol binding, with the following rank order of potencies: ( -Iisoproterenol > ( -)-epinephrine > ( -)-norepinephrine. p,-selective antagonist ICI-1 18 5 5 1 was approximately 3 log orders more potent than the p,-selective antagonist, betaxolol. These receptors were also coupled to an adenylate cyclase. These results suggest strongly that cultured human retinal pigment epithelial cells possess ,8,-adrenergic receptors. The potential significance of these findings with regard to retinal pigment epithelial functions is discussed herein.
Molecular and Cellular Biochemistry, 1996
The rat cA2-47 gene encodes the pharmacologically defined a2D-adrenergic receptor (C%D-AR) subtype. Previously, the expression of its mRNA was shown in bovine retina by amplification through the reverse transcription-polymerase chain reaction (RT-PCR) of a region corresponding to the rat c~2o-AR , amino acid (aa) residues 382-439, indicating the presence of this subtype in this neural tissue. In the present study, the structure of this gene has been probed and the encoded receptor subtype has been characterized in bovine retina and its photoreceptor cells. The deduced aa sequence of the two bovine gene fragments, aa residues 290-375 and aa residues 392-434, demonstrates 77% overall identity with the rat c%D-AR subtype and 80% overall identity with the mouse ot2D-AR. The receptor encoded by the bovine gene was expressed in the retina and its photoreceptors with the typical pharmacological characteristics established for the rat c/,2D-AR subtype: The receptor bound rauwolscine with a K D of 14 nM in the retina and with that of 19 nM in the photoreceptor cells; the binding association rate constant, k+l , for the ligand was 0.012 rain -~, the dissociation rate constant, k~, was 0.14 rain -~ and the half-time for dissociation was 5 rain. Oxymetazoline displaced the bound [3H]-rauwolscine with an ECs0 value of 85 nM, while SK & F 104078, and prazosin displaced the bound [3H]-rauwolscine with the respective ICs0 values of 900 nM and 3000 nM. The other CtE-AR subtypes -O%A-AR, Ct2B-AR, ~t2c-AR-were not detected in the retina and its photoreceptors. Thus, this study shows that the bovine ~2D" AR gene is a structural variant of the rat and mouse genes, that the bovine gene encodes the typical pharmacologically defined Ct2D-AR subtype, that this subtype is present in its exclusive form in the bovine retina and its photoreceptors, where it may be presynaptic in nature. (Mol Cell Biochem 159: 129-138, 1996)
Journal of Ocular Pharmacology and Therapeutics, 1989
Mplia-chyirotrypsiji-irKïuced ocular hypertension in albino rabbits is widely used as an experimental model to screen potential antiglaucoma drugs. The present study compares the intraocular pressure (IOP) response following the ocular application of single or repeated adrenergic agents in conscious albino and pigmented rabbits. A single instillation of clonidine was not as effective in lowering the IOP in pigmented hypertensive rabbit eyes as in albino hypertensive eyes. Similarly, betaxolol moderately lowered the IOP in albino rabbits but induced a slight response when pigmented rabbits were used as an experimental model. Twice-a-day applications of betaxolol in pigmented hypertensive eyes permitted an identical level of IOP decrease to be reached, as observed in a oneday study in albino rabbits, after at least 6 days of treatment. It has been suggested that the pigmented layers of the iris-ciliary body may act as sites for topically applied antiglaucoma drugs. Non-specific binding could explain in part the frequent discrepancy observed between the preclinical results obtained in albino hypertensive rabbit eyes and clinical results obtained in glaucomatous human eyes.
Acetylcholine in adrenergic terminals of the cat iris
The Journal of Physiology, 1970
1. Acetylcholine was bio-assayed in the normal cat iris, and also after selective sympathetic or parasympathetic denervation. Sympathetic denervation caused no significant change in the acetylcholine content of the cat iris, whereas selective parasympathetic denervation reduced the acetylcholine content below the level of delectability, which on the average was at about 5 % of the acetylcholine content of the normal iris. 2. It is concluded that if adrenergic terminals contain any acetylcholine, it is less than what is detectable with the methods available at present, and most certainly leds than 6 % of the acetylcholine content of cholinergic neurones. 3. On the basis of these and other recently obtained observations, the hypothesis of Burn & Rand (1965) of a cholinergic link in the adrenergic transmission is discussed. It is proposed that it is more reasonable to suppose an interaction between peripheral adrenergic and cholinergic terminals than to presume a cholinergic mechanism within adrenergic nerve fibres.
Beta adrenergic receptors on cultured human retinal pigment epithelium
Investigative Ophthalmology Amp Visual Science, 1990
Cultured fetal human retinal pigment epithelium (RPE) was grown on a permeable substrate and sealed in an Ussing chamber. The average electrical resistance (R) was 330 ohm-cm 2 , the average transcpithclial voltage (Ve) was 3.0 mV (apical side positive), and the average short circuit current (Isc) was 9.1 //A/cm 2. When these RPK preparations were exposed to isoprotercnol (a /8-adrenergic agonist), the Isc increased by 88%, R was reduced by 6%, and Ve increased by 85%. The effect of isoprotcrenol was blocked by propranolol (a /?-adrcnergic antagonist). When cultured human RPE was exposed to isoproterenol, intraccllular cyclic adenosine monophosphate (AMP) levels rose more than threefold. The effect of isoproterenol on cyclic AMP levels was blocked by propranolol. When the cultured RPK was exposed to dibutyryl cyclic AMP, both Ve and Isc rose by 47% with a time course similar to that which occurred when the cells were exposed to isoprotcrenol. Preparations treated with dibutyryl cyclic AMP did not respond to subsequently applied isoprotcrenol. These results indicate that cultured human RPE possesses a /3-adrenergic receptor and that stimulation of this receptor produces a change in cyclic AMP concentration which affects RPE electrical activity.
Albrecht von Graefes Archiv f�r Klinische und Experimentelle Ophthalmologie, 1969
The adrenergic retinal neurons were studied in cynomolgus and squirrel monkeys, cats, rabbits, guinea-pigs, rats and mice. In addition to what has previously been described, some very few adrenergie terminals were in all species observed to reach the horizontal cells. Also, except in the cynomolgus monkey, the inner plexiform layer contained three sublayers of adrenergic fibres. The two innermost layers were very scant in certain species. 9 By crushing the optic nerve, it was confirmed that it contains no adrenergic axons, except along vessels. Consequently, the adrenergie perikarya of the ganglion cell layer (the alloganglion cells) must be entirely intraretinal. Pharmacohistochemical experiments revealed that all cell types of the rat retina contain dopamine. A system of adrenergie neurons presumably containing noradrenaline was also detected. Their morphology was not obviously different from the neurons containing dopamine. Zusammen/assung. Die adrenergen retinalen Neurone wurden an Cynomolgusund Totenkopfaffen, Katzen, Kaninchen, Meerschweinchen, Ratten und M~usen studiert. Als Erg/~nzung zu friiheren Beschreibungen wurde beobachtet, dab bei allen untersuchten Arten nur sehr wenige der adrenergen Terminale die horizontalen Zellen erreiehten. Auch zeigte sich, dal], mi~ Ausnahme des Cynomolgusaffen, die irmere plexiforme Schicht drei Unterschichten enth/~lt, bestehend aus adrenergen Fasem. Die beiden innersten Schichten waren sehr spiirlich bei einigen der untersuchten Arten. Durch Zerquetschen des Opticus wurde best/itigt, dab dieser keine adrenergen Axone enth~lt, mit Ausnahme entlang der Gef~Be. Demnach mfissen die adrenergen Perikarya der Ganglienzellschicht (die AlloganglienzelIen) ausschlieBlich intraretinal liegen. Pharmakohisf~ochemische Experimente zeigten, daft alle Zelltypen der l~etina der Ratte Dopamin enthalten. Ein System yon adrenergen Neuronen, die wahrscheinlich Noradrenalin enthalten, wurde auch entdeckt. Ihre Morphologie unterschied sich nicht offensichtlich yon den dopaminenthaltenden lNeuronen. With the aid of the method evolved by FALCK and ItILLAI~r for inducing fluorescence in neurons containing certain catechol and indol derivatives, a system of adrenergic neurons has in recent years been detected in the retina of a wide variety of species ranging from teleosts to primates (