Contribution of different amine oxidases to the metabolism of dopamine in bovine retina.

The contribution of monoamine oxidase (MAO) A, MAO B and semicarbazide-sensitive amine oxidase (SSAO) to the metabolism of dopamine in the bovine retina was studied. These activities were present in the optic nerve, iris, choroid and bovine retina, but they were absent in the lens. SSAO activity towards dopamine was present in the choroid and the retina, but not in the iris or the optic nerve. The corresponding kinetic values for this substrate in the retina and the choroid showed higher affinity for MAO A (Km 271 and 197 microM, respectively) than for MAO B (Km 861 and 404 microM, respectively). This effect was counteracted by the higher Vmax value for MAO B resulting in the Vmax/Km ratio being similar for both cases. The absence of detectable SSAO activity towards dopamine in these last two tissues contrasts with the presence of that enzyme when benzylamine was studied as substrate. These results indicate that two different SSAO activities could be present in the bovine eye.     

Distribution of brimonidine into anterior and posterior tissues of monkey, rabbit, and rat eyes.

The objectives of the study were to evaluate the distribution of brimonidine (alpha2-adrenergic agonist) into anterior and posterior ocular tissues. Single or multiple doses of a 0.2 or 0.5% brimonidine tartrate solution were administered to one or both eyes of monkeys or to one eye of rabbits. Brimonidine was administered intraperitoneally to rats. After topical administration, [14C]brimonidine was rapidly absorbed into the cornea and conjunctiva and distributed throughout the eye. [14C]Radioactivity was higher and cleared more slowly in pigmented tissues (iris/ciliary body, choroid/retina, and optic nerve) than in nonpigmented tissues. Single and multiple dosing led to a similar drug distribution, with higher levels of brimonidine measured in pigmented tissues after multiple dosing. Most of the radioactivity extracted from ocular tissues represented unchanged brimonidine. In the rabbits and the monkey treated in only one eye, levels of radioactivity in the untreated eye were low, consistent with the low systemic levels and rapid drug clearance. Posterior ocular tissue concentrations of radioactivity exceeded systemic blood concentrations. The vitreous humor brimonidine concentrations in monkeys treated topically with 0.2% brimonidine tartrate was 82 +/- 45 nM. Vitreous levels in rabbits confirmed the penetration of brimonidine to the posterior segment. Similar concentrations of brimonidine (22 to 390 nM) were measured in the vitreous and retina of rats injected intraperitoneally with brimonidine. Both topically applied and systemically administered brimonidine reach the back of the eye at nanomolar concentrations sufficient to activate alpha2-adrenergic receptors. The brimonidine levels achieved at the retina are relevant for neuroprotection models.     

Histamine in retina, optic nerve, choroid and brain of albino and pigmented rabbits

Retina, optic nerve and various brain structures of both albino and pigmented rabbits contained histamine in the range of 40-400 ng/g tissue; choroid of both animal strains was characterized by several times higher amine content, i.e. 1.2-5.5 micrograms/g tissue. Among the studied structures the brain tissue was the only one able to synthesize substantially histamine from 1-histidine. Histological analysis of the ocular structures revealed the presence of mast cells only in the choroid of both albino and pigmented rabbits. Incubation of either the retina or optic nerve or choroid with compound 48/80 produced release of histamine only from the choroid.     

Comparative assessment of ocular tissue distribution of drug-related radioactivity after chronic oral administration of 14C-levofloxacin and 14C-chloroquine in pigmented rats

Fluoroquinolones have been reported to have a high affinity for melanin. The ocular tissue distribution and accumulation of radioactivity was compared after repeated oral administration of 14C-levofloxacin and 14C-chloroquine at daily doses of 20 mg (0.054 mmol) kg(-1) and 28 mg (0.054 mmol) kg(-1), respectively, in pigmented rats for 84 days. The mean serum level at 24 h following each dose of 14C-levofloxacin was almost constant in the range of 0.33-0.45 nmol equiv mL(-1) after the 14th dose and thereafter. The melanin-containing ocular tissues, such as iris ciliary body and stratum pigment chorioides sclera, showed a much higher concentration of radioactivity than other non-pigmented ocular tissues. The respective concentration in iris ciliary body and stratum pigment chorioides sclera after the 1st dose was 126.47 and 74.91 nmol equiv g(-1), and gradually increased with increasing dose number, reaching 1261.81 and 447.45 nmol equiv g(-1) after the 84th dose, which was ca. 10 and 6 times higher, respectively, than after the 1st dose. The mean serum level following each dose of 14C-chloroquine was almost constant in the range 0.51-0.87 nmol equiv mL(-1) after the 7th dose and thereafter. The respective concentration in iris ciliary body and stratum pigment chorioides sclera after the 1st dose was 572.10 and 709.41 nmol equiv g(-1), and gradually increased with increasing dose number, reaching 33 317.92 and 12 322.90 nmol equiv g(-1) after the 84th dose, which was ca. 58 and 17 times higher, respectively, than after the 1st dose. The concentration in aqueous humour, cornea, lens, vitreous body and retina after the 84th dose was 1.84, 6.33, 0.48, 5.60 and 11.42 nmol equiv g(-1) for 14C-levofloxacin and 18.84, 264.99, 27.26, 158.43 and 1020.89 nmol equiv g(-1) for 14C-chloroquine (ca. 10, 42, 57, 28 and 89 times higher, respectively, than for 14C-levofloxacin). Especially, the concentration in the retina was markedly higher after 14C-chloroquine administration than after 14C-levofloxacin administration. The concentration and the extent of accumulation of radioactivity not only in melanin-containing ocular tissues but also in other non-pigmented ocular tissues, such as retina, after chronic oral administration of 14C-levofloxacin once daily for 84 days were much lower than those after multiple dosing with 14C-chloroquine under the same conditions. These results indicate that levofloxacin would have a much lower risk for ocular toxicity than chloroquine after chronic dosing.     

Über das Vorkommen von Katecholaminen und von 3,4-Dihydroxyphenylalanin (Dopa) im Auge

Summary 1. The occurrence and distribution of catecholamines in the ocular tissue of the calf has been investigated by a fluorimetric method.2. In the lens, in the vitreous humour and in the retina (without the pigment-epithelium) no significant amounts of noradrenaline have been detected. In the chorioideal layer, isolated together with the retinal pigment-epithelium, noradrenaline was found in concentrations from 0.01 to 0.15 g/g wet tissue. The noradrenaline concentration in the iris was in the range of 0.19–0.66 g/g wet tissue.3. The adrenaline concentration, as estimated in one sample of iris tissue, was as low as 0.05 g/g.4. Another fluorescent catechol compound was found in the iris as well as in the chorioideal layer (isolated together with the retinal pigment-epithelium).5. This catechol compound was found to be 3.4-dihydroxyphenylalanine (dopa), as identified by twodimensional paper chromatography, separation on ion-exchange and Al₂O₃ columns, colour reactions, fluorescent behaviour and by enzymatic decarboxylation to dopamine.6. The dopa concentration in these tissues was in the range of 1.06 to 4.34 g/g wet tissue, or even higher, as might be concluded from the experimental data.7. The occurrence of dopa in the ocular tissue might possibly be connected with the metabolism of melanin.

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Ein Teil der Ergebnisse wurde auf der 4. Frühjahrstagung der Deutschen Pharmakologischen Gesellschaft in Mainz vorgetragen [Naunyn-Schmiedebergs Arch. exp. Path. Pharmak. 246, 12 (1963)].     

Comparison of post-junctional α-adrenoceptors in iris dilator muscle of humans, and albino and pigmented rabbits

The relative potency of -adrenoceptor agonists and the dissociation constants of competitive antagonists were studied to characterize the post junctional -adrenoceptor of the human iris dilator muscle. The data obtained from human iris dilator tissue was compared to that from rabbit. The iris dilator muscle was mounted in an organ bath and tension changes were recorded. (–)-Norepinephrine, (–)-phenylephrine (PE), oxymetazoline and p-aminoclonidine caused contractile responses in albino rabbit, pigmented rabbit and human iris dilator muscle in a concentration-dependent manner. The imidazoline molecules were partial agonists. In rabbit iris dilator, desensitization occurred to repeated oxymetazoline application at an interval of 1 h but recovery to the agonist activity was complete in about 3 h. Exposure to cocaine (10 mol/l), hydrocortisone (100 mol/l) and U-0521, a catechol-O-methyltransferase inhibitor (100 pmol/l), significantly potentiated the response to norepinephrine by 92-, 32- and 7 fold in iris dilator tissue of albino rabbit, pigmented rabbit and human, respectively. After block of uptake₁ and uptake₂, the EC₅₀ values of norepinephrine in the albino rabbit, pigmented rabbit and human iris dilator did not differ and ranged from 99 to 195 nmol/l. Small but significant potentiation by uptake blockers was also observed in the responses to PE in the albino rabbit or pigmented rabbit iris dilator. The average maximum tension induced by 100 mol/l PE was 96 ± 11 mg (n = 10), 197 ± 11 mg (n = 11), 45 ± 5 mg (n = 27) in albino rabbit, pigmented rabbit and human iris dilator, respectively.In human iris dilator, the responses to PE were competitively antagonized by prazosin, 5-methylurapidil and phentolamine with apparent pKB values of 7.3, 6.6 and 7.5, respectively. The pK _B values of the prazosin-PE interaction in iris dilator of albino and pigmented rabbit were 8.6 and 6.4, respectively.These results suggest that the post-junctional -adrenoceptors in iris dilator may be similar to that in pigmented rabbit iris. The -adrenoceptor of the human or pigmented rabbit iris dilator may be characterized as _1L-adrenoceptor subtype. The -adrenoceptor of albino rabbit iris dilator appears to be a high affinity subtype.Furthermore, albino rabbit may not be the best strain for the drug research which is relevant to human ocular therapeutics.     

Improvement of ocular blood flow with dopamine antagonists on ocular-hypertensive rabbit eyes.

The eyedrops of the ocular-hypotensive dopamine antagonists, trifluperidol, moperone, lenperone, and spiperone, were instilled into an ocular-hypertensive rabbit eye. The blood flows in the choroid, retina, iris root-ciliary body, and iris were measured with colored microspheres at various time periods. It was found that all these dopamine antagonists, at a concentration of 0.5%, increased the blood flow in all eye tissues. Dopamine, at a concentration of 3%, produced a biphasic action by decreasing the blood flow initially at 30 min, then increasing it at 120 min and thereafter. But 1.5% dopamine produced a monophasic action which increased the blood flow after 180 min. Since dopamine antagonists are not cholinergics or adrenolytics, they are not supposed to produce the side effects induced by pilocarpine or timolol. It is hoped that they can become satisfactory drugs for glaucoma and ocular hypertension.     

白芍总苷对葡萄膜炎模型大鼠的保护作用

目的:研究白芍总苷对葡萄膜炎模型大鼠的保护作用。方法:于大鼠足底部注射伤寒杆菌内毒素(LPS)200μg以复制大鼠葡萄膜炎模型。32只SD大鼠随机均分为正常对照(等容生理盐水)组、模型(对容生理盐水)组、白芍总苷(4.8 g/kg,每隔6 h给药1次,共3次)组、醋酸泼尼松(0.05 g/kg,给药1次)组,复制模型前1 h开始灌胃给药。用裂隙灯显微镜观察大鼠临床改变,通过病理学检查观察大鼠眼部组织学改变。结果:正常对照组未见炎症发生;与正常对照组比较,模型组炎症临床评分升高,差异有统计学意义(P<0.05),大鼠虹膜、睫状体、视网膜与玻璃体腔炎症细胞浸润程度明显加剧;与模型组比较,白芍总苷组炎症临床评分降低,差异有统计学意义(P<0.05),大鼠虹膜、睫状体、视网膜与玻璃体腔炎症细胞浸润程度明显改善。结论:白芍总苷对LPS诱导的葡萄膜炎大鼠有一定保护作用。     

Comparative study of phase II biotransformation in rabbit ocular tissues.

To assess the biotransformational capability of ocular tissues in the rabbit, representative phase II enzymes were assayed in five tissues from the eye, and in the liver, kidney, and intestine. Within the eye, the iris/ciliary body exhibited the highest glutathione S-transferase activity, whereas the cornea possessed the highest specific activities for N-acetyl-, sulfo-, and UDP-glucuronosyl-transferases. Cornea, iris/ciliary body, choroid, and retina exhibited significant activities of p-aminobenzoic acid N-acetyltransferase, 2-naphthol sulfotransferase, and 1-chloro-2,4-dinitrobenzene glutathione S-transferase. Despite its size and protein content, lens displayed little or no biotransformational activity. Only the iris/ciliary body conjugated sulfobromophthalein with glutathione. UDP-glucuronsyltransferase activity varied depending on tested substrates and tissues. When compared to liver, kidney, or intestine, N-acetyltransferase activity in the iris/ciliary body nearly matched the rate measured in kidney, glutathione S-transferase activity in cornea and iris/ciliary body was nearly 70 and 89%, respectively, of the rate in intestine, and corneal sulfotransferase activity was greater than that in kidney. These data suggest that biotransformation pathways are present in the eye, and particularly in ocular tissues having adequate blood supply or interfacing with the external environment.     

GABAergic modulation of D-1 dopamine receptor-mediated 3H-acetylcholine release from rabbit retina

Summary Dopamine evokes calcium-dependent release of ³H-acetylcholine from superfused rabbit retina labeled in vitro with ³H-choline, through activation of a D-1 dopamine receptor. This study investigates the activation of this receptor by endogenous dopamine and the modulation of the spontaneous and dopamine-evoked release of ³H-acetylcholine from rabbit retina labeled with ³H-choline by GABAergic agonists and antagonists. Endogenous dopamine, released from dopaminergic amacrine neurons by the indirect amines tyramine or D-amphetamine evoked the calcium-dependent release of ³H-acetylcholine from rabbit retina. The release of ³H-acetylcholine elicited by tyramine (10 M) or D-amphetamine (10 M) was attenuated by the selective D-1 antagonist SCH 23390 (0.1 M) and by the dopamine uptake inhibitor nomifensine (3 M). At concentrations of 1 mM and 1 M respectively, GABA and muscimol inhibited the spontaneous release of tritium from rabbit retina labeled in vitro with ³H-choline. Picrotoxin and bicuculline (10 M) increased the spontaneous release of tritium. GABA and the GABA agonist muscimol (0.01–100 M) inhibited in a concentration-dependent manner the release of ³H-acetylcholine elicited by 100 M dopamine with IC₅₀ values of 4.5 M and 0.02 M respectively. The inhibition of dopamine-evoked ³H-acetylcholine release by GABA (10 M) and muscimol (0.1 M) was antagonized by the GABA antagonists bicuculline and picrotoxin. Picrotoxin and bicuculline (10 M) increased the spontaneous release of tritium, and potentiated the release of ³H-acetylcholine evoked by 100 M dopamine consistant with a tonic, inhibitory GABAergic input to the cholinergic amacrine neurons in rabbit retina. Dopamine-evoked acetylcholine release in rabbit retina may be of physiological importance as D-1 dopamine receptor-mediated increases in ³H-acetylcholine release from rabbit retina can be elicited by endogenous dopamine. In addition, activation of GABA receptor sites modulates the spontaneous and dopamine-evoked acetylcholine release from rabbit retina. Send offprint requests to M. L. Dubocovich at the above address