Ocular development and aging. 1. Corneal endothelial changes in cats and in free-ranging and caged rhesus monkeys.

The endothelium of the cornea is said to retain its cellular proliferative capacity in rabbits but not in cats or primates. The present study was undertaken to determine whether the decline in cell density with increasing age is shared by all species that presumably lose their corneal endothelial-cell proliferative capacity and to determine whether in primates age-dependent changes in the endothelium are influenced by such environmental factors as light intensity and temperature. We found that in cats a very rapid decline in endothelial cell density during the first 10 months post partum was followed by a slower decline between 10 and 12 months, corresponding to a rapid increase followed by a slower rate of increase in corneal surface area. Between 1- and 14 years of age, a further decline in endothelial-cell density did not occur, and no endothelial pleomorphism or corneal guttata was observed among the 44 cats studied in this age group. In rhesus monkeys, an approximate 20% decline in cell density between 1 and 6 years of age, representing a period of ocular growth, was followed by a continuing decline in endothelial cell density at a slower rate of 0.7 to 0.8% per year throughout the rest of the lifespan, despite the fact that there was no evidence for further increase in globe size during this period of adulthood and aging. This rate of decline is similar to that described in the human corneal endothelium and was found to be the same in free-ranging animals continually exposed to high levels of solar radiation and in animals caged under artificial room light of much lower intensity and a year-round temperature of 21 +/- 1 degrees C. However, the animals maintained indoors showed a much higher frequency of endothelial pleomorphism than the free-ranging animals. These differences in age-dependent changes between feline and primate corneal endothelium and between primates living under grossly different environmental conditions are discussed.

Eicosanoids as a new class of ocular hypotensive agents. 1. The apparent therapeutic advantages of derived prostaglandins of the A and B type as compared with primary prostaglandins of the E, F and D type.

The classic primary prostaglandins (PGs), as well as some of their analogs and derivatives, are potent ocular hypotensive agents. The present studies show that A and B PGs, which are derived from PGs of the E type by dehydration and isomerization, have a much greater ocular hypotensive potency than the primary PGs of the E, F or D type. A single application of 5 micrograms of PGA2 to the cat eye in a 25-microliters volume of aqueous vehicle solution yielded a greater and more prolonged ocular hypotensive effect than as much as 100 micrograms of topically applied PGF2 alpha. As little as 1 microgram of PGA2 had a significant ocular hypotensive effect that was enhanced by three or more consecutive daily applications of the same dose. This IOP reduction, which remained significant for several days after the last of 10 daily treatments, was not associated with biomicroscopically detectable flare or invasion of the anterior chamber by cells. Although PGF2 alpha and, to a much lesser extent, PGE2 have a miotic effect in cats, PGs of the A and B type did not cause significant miosis even at doses 50- to 100-fold greater than the minimum dose required to yield significant ocular hypotension. PGA2 retained its ocular hypotensive potency when stored in an aqueous solution at room temperature for four months. The conjunctival hyperemia caused by 5 micrograms or 10 micrograms of A or B type PGs on rabbit eyes was milder and shorter in duration than that caused by the same doses of PGE2 or PGF2 alpha. These findings suggest that derived PGs, especially PGs of the A type, may have a therapeutic advantage over primary PGs for the treatment of ocular hypertension and glaucoma.

The ocular pharmacokinetics of eicosanoids and their derivatives. 1. Comparison of ocular eicosanoid penetration and distribution following the topical application of PGF2 alpha, PGF2 alpha-1-methyl ester, and PGF2 alpha-1-isopropyl ester.

These experiments were undertaken to determine whether the increased ocular hypotensive potency of topically applied prostaglandin (PG) PGF2 alpha esters, as compared with that of PGF2 alpha free acid, can be accounted for by increased penetration of the eicosanoid moiety of the esterified PG into the eye. One hour after the topical application of [3H]PGF2 alpha-1-methyl ester (ME) in peanut oil, the 3H activities in the cornea, aqueous humor, and ciliary body of the rabbit eye were 32-, 22-, and 8-fold higher, respectively, than they were following the topical application of [3H]PGF2 alpha free acid. 3H activity during the first 3 hr declined rapidly in the cornea and more slowly in the aqueous humor, but remained essentially constant in the ciliary body for up to 6 hr, declining rapidly only between 6- and 24 hr. 3H activity in eyes that received [3H]PGF2 alpha ME was also several-fold higher in the anterior sclera and iris than in eyes that were treated with [3H]PGF2 alpha free acid, but this difference was much smaller in the conjunctiva. At 1 hr, most of the 3H activity in the aqueous humor was associated with PGF2 alpha, as determined by chromatography, but at 2- and 3 hr other peaks, presumably reflecting metabolites of PGF2 alpha, became apparent. The penetration and intraocular distribution of 3H activity was similar when [3H]PGF2 alpha ME was applied to the eye in normal saline rather than in peanut oil or when the isopropyl rather than the methyl ester of PGF2 alpha was used. These studies indicate that esterification of the carboxyl group of PGF2 alpha greatly enhances the penetration of the PGF2 alpha moiety into the eye and suggests that effective de-esterification of the PGF2 alpha ester occurs in the cornea, resulting in the delivery of PGF2 alpha free acid into the aqueous humor. It is concluded that topically applied PG esters act as pro-drugs and that the increased ocular penetration of these esters may account for the previously reported increase in their ocular hypotensive potency as compared to that of PG free acid or salts.

Ocular trace metal kinetics and toxicology. I. The distribution of intravitreally injected 67Cu++ within intraocular compartments and its loss from the globe.

Radioactive copper (67Cu++) was injected into the center of the vitreous body of rabbits. The relatively rapid initial loss of 67Cu from the vitreous was associated with its accumulation in intraocular tissues. At 24 hr, 20% of the injected 67Cu was found in the retina, representing the highest 67Cu concentration among all ocular tissues, and this high 67Cu concentration was maintained in this tissue throughout the 10-day observation period. Significant amounts of 67Cu were not detected in the aqueous humor at any time. About one-half of the injected 67Cu was lost from the whole globe in 5 days, but the remaining Cu was retained in the globe during the next 5 days. Eyes that received a large dose of CuSO4 in addition to the tracer showed decreased 67Cu activity in the retina and a slight increase in the aqueous humor. Endotoxin-induced ocular inflammation decreased the rate of 67Cu loss from the vitreous, reduced its accumulation by the retina, and increased 67Cu entry into the aqueous humor. It is concluded that 67Cu is retained in the vitreous and the globe due to its binding by, and/or uptake into, intraocular tissues, especially the retina. Cu does not effectively enter the anterior chamber from the vitreous, apparently due to its effective removal by the ciliary processes, thus ruling out the possibility of identifying the existence of Cu-containing intraocular foreign bodies in the posterior segment of the eye by analysis of Cu in the aqueous humor.

Noninvasive observations on eyes of cats after long-term maintenance of reduced intraocular pressure by topical application of prostaglandin E2.

Daily or twice daily prostaglandin E2 (PGE2) application to cat eyes was shown to maintain a reduced intraocular pressure (IOP) for several months without causing substantial flare or cellular response. We report now on detailed ophthalmic examinations performed on these cats after 5-9 months of such treatment (ie, after 150 to 250 unilateral PGE2 applications; 100 micrograms/treatment per eye). A comparison of the treated and contralateral control eyes revealed no differences in the axial length of ocular compartments, in the biomicroscopic appearance of the lens, vitreous, retina, or optic nerve head, in the rate of light-induced pupillary constriction or in the wave form of the electroretinogram. The cell density of the corneal endothelium was not decreased, but the endothelial surface did contain a few small "dark spots." A slight iridial heterochromia was generally apparent. In three of the cats PGE2 application had a sialagogic effect that became a conditioned reflex. Cats tended to keep their lids closed after each treatment; lid closure was more prolonged in the PGE2-treated eye than in the contralateral eye that received the same volume (50 microliters) of vehicle solution. It is concluded that daily treatment with PGE2, in doses sufficient to cause a maintained reduction in IOP, does have some side effects. However, none of these side effects are of sufficient importance to exclude the use of eicosanoids as potential anti-glaucoma agents.

The impermeability of the basic cell membrane to thromboxane-B2' prostacyclin and 6-keto-PGF 1 alpha.

Washed rabbit red blood cells (RBCs) were suspended in electrolyte solution containing 3H-labeled prostacyclin (PGI2), thromboxane (TxB2) or 6-keto-PGF 1 alpha and 14C-labeled sucrose or thiourea. following 1 to 30 min incubation with 14C-sucrose, 3H-TxB2 or 3H-6-keto-PGF 1 alpha, the 14C or 3H space of packed RBCs remained essentially constant, yielding mean values (+/- S.E.) for all time periods of 6.1 +/- 0.3, 9.5 +/- 0.5 and 6.5 +/- 0.4%, respectively. After 1 min of incubation at 4 degrees or 23 degrees C at a pH of 7.4 or 8.5 with trace amounts (10(-9)M) of 3H-PGI2 or in the presence of added PGI2 (10(-5)M) or ethacrynic acid (1.6 x 10(-4)M), the apparent PGI2 space of packed RBCs ranged from 16 to 27%, decreasing to about 7% by 30 min. When RBCs were resuspended in fresh 3H-PGI2 every 5 min, their 3H content increased very slowly (apparent PGI2 space less than 40% at 30 min) as compared to thiourea (distribution space greater than 80% within 5 min). Over 90% of this 3H activity was lost from the RBCs in less than 2 min during elution at 4 degrees or 23 degrees C. It is concluded that RBC membranes and thus, presumably, the basic cell membrane in general, is not fundamentally permeable to PGI2, 6-keto-PGF 1 alpha or TxB2. Hence, the effective entry of these cyclooxygenase products into some cells or their passage across tight-junctional capillaries or epithelial membranes must require facilitated or active transport processes as was shown to be the case for E, F and A PGs. This implies that the distribution, pharmacological action and metabolism of these and presumably all related cyclooxygenase products are selective rather than unrestricted.

The penetration of exogenous prostaglandin and arachidonic acid into, and their distribution within, the mammalian eye.

The distribution of 3H and 14C activities in intraocular fluids and tissues was studied after topical application of 3H-prostaglandin F2 alpha (3H-PGF2 alpha) and/or 14C-arachidonic acid (14C-AA) to rabbit eyes; intravenous (i.v.) infusion of 3H-PGF2 alpha into rats; and after incubation of rat or rabbit globes in media containing these and other tracers. Thirty min after topical application of 14C-AA, the order of distribution of 14C per unit of tissue weight was cornea much greater than sclera congruent to ciliary body congruent to iris congruent to aqueous greater than retina-choroid greater than vitreous greater than lens. The distribution of 3H was sclera greater than cornea greater than retina-choroid congruent to ciliary body greater than aqueous greater than vitreous (lens congruent to 0; iris congruent to 0). After i.v. infusion of 14C-sucrose and 3H-PGF2 alpha into rats for 1 to 5 min, the globe/blood ratio of 3H was significantly lower than that of 14C. When isolated rat globes were incubated, the order of tracer uptake into the whole globe was AA greater than thiourea greater than PGF2 alpha greater than sucrose, while the order of entry into the aqueous was thiourea greater than sucrose greater than AA greater than -PGF2 alpha. The isolated rabbit cornea accumulated large amounts of 14C-AA which was not readily elutable and much smaller amounts of 3H-PGF2 alpha which was readily elutable. It is concluded that the sclera is highly permeable to PGF2 alpha, but that the cornea is an effective permeability barrier to this, and presumably all other PGs. The passage of AA through the outer coats of the globe is limited by its chemical incorporation into the cornea, sclera and conjunctiva. Thus, inhibition of the adverse intraocular effects of topically applied AA by topically applied drugs may only reflect the ability of these drugs to inhibit the synthesizing capacity of the superficial layers of the globe rather than that of the anterior uvea and other intraocular tissues.

The mechanism of peptidergic miosis. I. The structural basis of miotic potency among biologically active polypeptides.

The miotic potency of eleven naturally occurring polypeptides and poly-DL-alanine was tested on the isolated anterior segment of hooded rat and golden hamster eyes in order to gain information on the chemical nature of the peptides or classes of peptides which may be regarded as possible mediators of the miotic response of the mammalian eye to chemical irritants or mechanical or surgical trauma. Substance P, P-octapeptide, physalaemin and eledoisin, and eledoisin related peptide were found to be potent miotics on these preparations, yielding ED50 values in the range of 8 to 95 nM and complete pupillary constriction in the range of 100 to 1000 nM. This is comparable to the miotic potency of carbachol or serotonin. All of these peptides share a C-terminal sequence Phe-X-Gly-Leu-Met-NH2 (where "X" is a variable amino acid) which is characteristic of all tachykinins. Six other biologically active peptides or the synthetic poly-DL-alanine had no measureable miotic effect on these preparations up to a final concentration of 1000 to 10,000 nM. The miotic effect of SP on the rat iris was effectively, but not completely antagonized by the presence of 10(-7)M atropine sulfate in the incubation medium. Preliminary results indicate that the miotic effect of physalaemin, eledoisin and eledoisin related peptide on the anterior segment of rat eyes is similarly antagonized by atropine.

Dependence of pulmonary prostaglandin metabolism on carrier-mediated transport processes.

Inhibitors of prostaglandin (PG) transport (probenecid, indomethacin, or bromcresol green) were found to eliminate the difference between the pulmonary transit time of 3H and 14C when [3H]PGF2alpha and E114C]sucrose were injected as a single intra-arterial bolus into the isolated perfused rat lung. Similar results were obtained with PGE1. The transit time of [3H]PGA1 was not significantly different from that of [14C]sucrose even in the absence of an inhibitor. These inhibitors increased the amount of [3H]PGF2alpha or [3H]PGE1 and decreased the amount of [3H]PG metabolites found in the venous effluent: these agents also inhibited the pulmonary metabolism of continously infused, nonradioactive PGF2alpha. One of the three inhibitors, bromcresol green, was shown not to be an effective inhibitor of PG metabolism in cell-free preparations of rat lung homogenates. These results indicated that under normal conditions, PG's are rapidly transported into intracellular compartment(s) where they are metabolized. Inhibition of this transport process prevents rapid access of PG's to the cytoplasmic enzymes and therefore inhibits pulmonary PG metabolism. This implies that inhibitors of PG transport, including anti-inflammatory organic acids, and some PG antagonists, metabolites, and analogues, can be expected to inhibit the pulmonary metabolism of PG's and thus could potentiate the systemic effects endogenous or exogenous PG's.

Impermeability of rabbit erythrocytes to prostaglandins.

Washed rabbit erythrocytes were suspended in Tris-electrolyte buffer containing [3H]prostaglandin (PG) E1, F1alpha, F2alpha, or A1 and one 14C-labeled compound such as sucrose. After up to 24 h of incubation, aliquots of centrifuged, packed cells and supernatant were oxidized and the 3H and 14C samples were counted. The mean sucrose space of the packed cell was 7.4%. After 1 min the E1, F1alpha, and F2alpha spaces were 16, 15.4, and 10.0%, respectively, and showed no increase even after 24 h of incubation at either 23 or 5 degrees C. At 23 degrees C the initial (0.5 min) PGA1 and thiourea spaces were 94 and 75%, respectively, whence the PGA1, but not the thiourea, space declined, reaching 30% at 4 h. The large initial uptake of PGA1 was eliminated at 5 degrees C, while it was accentuated at pH 6.8 or at a PGA1, concentration of 10(-3) M. 14C-Labeled arachidonic, octanoic, and other non-PG fatty acids gave apparent distribution spaces of 140-300%. These results show that PG's can partition into rabbit erythrocyte membranes, but the intracellular volume of the erythrocytes is not freely accessible to these autacoids. The implications of the finding that some cell membranes are impermeable to prostaglandins are discussed.