Antioxidant and anticataractogenic effects of topical captopril in diquat-induced cataract in rabbits.

1-[(2s)-3-Mercapto-2-methylpropionyl]-L-proline (captopril), an antihypertensive and free radical scavenger, protected the rabbit lens from peroxidative and oxidative damage induced by 1 mM diquat in vitro. To evaluate the anticataract efficacy of captopril, an experimental group of five rabbits was treated with topical captopril (1% in 0.15 M NaCl, w/v), and 50 microliters was instilled onto both eyes four times a day for a total of 8 weeks. Following the same procedure, the eyes of five rabbits were treated with topical 0.15 M NaCl as a control for captopril treatment. At the end of the first week of treatment, a single intravitreal dose of 120 nmole diquat in 30 microliters of 0.15 M NaCl was injected into the right eye of each rabbit of both the groups. As a control for intravitreal diquat injection, the left eye of all the rabbits were injected with the diluent, 30 microliters per eye. The intravitreal diquat or its diluent injection was only for one time. From slit-lamp biomicroscopic observation of the diquat-injected right eyes, the anticataract effect of captopril in the treatment group was indicated by the finding that in four of five rabbits the cataract did not advance; whereas in four of five rabbits treated with the diluent the cataract progressed to grade 3. The lenses in the diluent-injected control left eyes of the rabbits treated with the captopril or diluent were normal. However, since the number of animals used for the in vivo studies was few, further confirmation of the anticataract effect of captopril is necessary. In diquat-injected right eyes of animals treated with captopril, the integrated rate of O2- production was about 50% less (p less than .001) in the aqueous humor, vitreous humor, and lens, compared with O2-, 33.49 +/- 2.26 microM (mean +/- SEM) in the aqueous humor, 17.12 +/- 0.75 microM in the vitreous humor, and 31.44 +/- 1.29 nmole/g wet weight in the lens of the diquat-injected right eyes treated with the diluent. Similar significant (p less than .01) differences in the production of .OH and H2O2 in eye tissues were also observed.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of dipyridamole on prostaglandin-induced ocular hypertension in rabbits.

The effect of intraperitoneal injections of dipyridamole on the elevations of intraocular pressure and anterior chamber aqueous humor protein produced by topical application of prostaglandin E2 was studied in rabbits. Pretreatment with 100 mg/kg dipyridamole inhibited the prostaglandin E2--induced ocular hypertension and rise of aqueous humor protein. Systemic administration of dipyridamole did not alter the responses of the eye to instilled nitrogen mustard. Topical instillation of dipyridamole was ineffective. Dipyridamole, a clinically available drug, may be useful in the treatment of ocular inflammatory disease.

Effect of imidazole on ionophore-induced ocular hypertension.

The effect of intraperitoneal injections of imidazole on the elevation of intraocular pressure produced by the topical application of the cation ionophores A23187 or X537A was studied in rabbits. Pretreatment with 200 mg./kg. imidazole completely inhibited the ocular hypertension induced by 1.0 percent A23187. The elevation of intraocular pressure produced by 0.5 percent X537A was not blocked by pretreatment with imidazole.

Inhibition of active transport of chloride and sodium by vanadate in the cornea.

Vanadate, an inhibitor of (Na+ + K+)ATPase, also inhibits active transepithelial Na+ and Cl- transport (measured as short-circuit current) in the isolated cornea in concentrations of 10(-4)M to 10(-3)M. The disulfonic stilbene, DIDS, does not affect the short-circuit current per se, but it prevents the inhibitory effect of vanadate on Na+ and Cl- transport. Since vanadate acts in other systems from the cytoplasmic side, it is postulated that DIDS interferes with vanadate penetration into the epithelial cells. These results also strengthen the notion of a Na-Cl co-transport system in the corneal epithelium.

Phorbol ester: effect on intraocular pressure, adenylate cyclase, and protein kinase in the rabbit eye.

Protein kinase C was identified as a major protein kinase enzyme activity in rabbit ciliary processes. Phorbol myristate acetate (4 beta-PMA) in the presence of Ca2+ activated protein kinase C but did not directly affect the cyclic AMP-dependent protein kinase enzyme isolated from ciliary processes. To elucidate possible roles of protein kinase C, PMA was injected intravitreally into rabbit eyes. Fifty pmoles of PMA produced approximately a 40% decrease of the intraocular pressure relative to the control eye lasting for more than 72 hr. A reduction of intraocular pressure was still elicited by this dose of PMA in animals pretreated with systemic indomethacin given to suppress a possible inflammatory response. The biologically inactive analogue, 4 alpha-phorbol didecanoate (100 pmoles/eye) had no significant effect on intraocular pressure. In vivo and in vitro treatment with PMA had no significant effect on adenylate cyclase in ciliary process membranes assayed in vitro. However, protein kinase C isolated from rat brain, when added together with cofactors to membranes in vitro, augmented adenylate cyclase activation by isoproterenol, vasoactive intestinal peptide and aluminum fluoride. A slight increase in the basal activity and in the forskolin response was not statistically significant. The effect of protein kinase C to increase responsiveness of ciliary process adenylate cyclase was totally dependent on the presence of Ca2+ and was augmented by addition of PMA. These findings indicate modulation of adenylate cyclase activity by protein kinase C acting at the level of the G-proteins and suggest a possible role for this enzyme in water and electrolyte transport in the ciliary processes.

Calcium-dependent phosphorylation of proteins in rabbit ciliary processes.

Calcium-dependent phosphorylation of endogenous substrate proteins in albino rabbit ciliary processes was studied by SDS-polyacrylamide gel electrophoresis and autoradiography. In the soluble fraction, a modest augmentation of phosphorylation was observed by Ca2+ alone and together with the additional activators, calmodulin (CAM) or phorbol myristate acetate (PMA). However, there was a greater enhancement of protein phosphorylation by Ca2+ and activators in the particulate fraction. The degree of Ca2+-CAM-dependent protein phosphorylation was greater than that of Ca2+-PMA-dependent phosphorylation. Endogenous substrate proteins for Ca2+-CAM-dependent protein kinases had apparent molecular sizes of 205,170,150,130,77,58,40,32 and 18 kDa. Phosphorylation of the 58 kDa protein band was strongest. This protein was identified as vimentin on the basis of its behavior with Triton-X100 treatment, and by Western blotting using anti-vimentin antibody. Endogenous substrates of protein kinase C (Ca2+-PMA-dependent) were located at 87 kDa and possibly in the 56 and 54 kDa protein bands. A 50 kDa protein was found to be phosphorylated in the presence of Ca2+ alone, and was not affected by the presence of other activators (CAM or PMA). A Ca2+-dependent dephosphorylation of a 43 kDa protein was observed, and some proteins rapidly phosphorylated by Ca2+-CAM kinase were also relatively quickly dephosphorylated at incubation times greater than 1 min.

Topical vanadate lowers intraocular pressure in rabbits.

In unanesthetized rabbits the topical application of vanadate lowered intraocular pressure. Tonographic outflow facility and episcleral venous pressure were unaltered by topical vanadate. As estimated from the tonographic data, aqueous humor flow was reduced by approximately 30%. Posterior chamber aqueous humor ascorbate increased in the eye receiving topical vanadate, and this was compatible with a decreased rate of aqueous humor flow. Topical vanadate did not alter anterior chamber aqueous humor protein or cyclic AMP. In five monkeys intraocular pressure was also significantly reduced by topical vanadate.

Effect of prostaglandin F2 alpha on aqueous humor dynamics of rabbit, cat, and monkey.

Topical administration of prostaglandin F2 alpha (PGF2 alpha) produced a reduction in intraocular pressure in eyes of rabbits, cats, and cynomolgus monkeys. In rabbit eyes at 5 or 6 hr, 50 micrograms, 100 micrograms, or 250 micrograms of PGF2 alpha caused a significant intraocular pressure reduction with a small miotic effect. Treatment with 500 micrograms, 750 micrograms, or 1000 micrograms of PGF2 alpha lowered intraocular pressure significantly in cat eyes for at least 24 hr with the development of profound pupillary constriction. Administration of 500 micrograms, 750 micrograms, or 1000 micrograms of PGF2 alpha produced a significant reduction of intraocular pressure in monkey eyes lasting at least 24 hr, with an initial hypertensive phase and a small decrease in pupillary diameter in the treated eyes. Tonography revealed an increased facility of outflow simultaneous with the reduction of intraocular pressure in the eyes of cats and monkeys. These increases of outflow facility could not explain completely the reductions in intraocular pressure. The aqueous humor flow measured by fluorophotometry was unaltered in both species, and possible reasons for this finding are discussed. Anterior chamber aqueous humor protein was significantly higher in cat eyes topically treated with 750 micrograms of PGF2 alpha than in the diluent-treated fellow eyes.

Cyclic nucleotide-dependent phosphorylation of proteins in rabbit ciliary processes.

Cyclic nucleotide-dependent protein phosphorylation in albino rabbit ciliary processes was studied in particulate and soluble fractions of the tissue by the technique of SDS-polyacrylamide gel electrophoresis and autoradiography. In the presence of gamma-32P-ATP, the soluble fraction showed increased phosphorylation of proteins of 200, 32 and 16 kDa molecular weight when 10 microM cAMP was added. Protein phosphorylation increased with time up to 5 min. No significant augmentation of phosphorylation was observed in the presence of 10 microM cGMP compared to control. In the particulate fraction, proteins with molecular weights of 200, 160, 105, 72, 58, 32 and 16 kDa showed increased phosphorylation in the presence of 10 microM cAMP. Phosphorylation caused by the addition of cAMP was maximal between 30 sec and 1 min for the particulate membrane fraction, but with longer incubation times the incorporation of phosphate residues decreased. The same molecular weight proteins of the membrane fraction that were phosphorylated in a cAMP-dependent manner were phosphorylated in the absence of exogenous cAMP by addition of either the catalytic subunit of cAMP-dependent protein kinase or activators of membrane-bound adenylate cyclase such as l-isoproterenol, vasoactive intestinal peptide, aluminum fluoride or forskolin. A cAMP-dependent dephosphorylation of a 56 kDa protein was observed in the membrane fraction. Cyclic GMP did not cause observable changes in the pattern of protein phosphorylation in the particulate fraction of rabbit ciliary processes.(ABSTRACT TRUNCATED AT 250 WORDS)

Electrical parameters of the isolated monkey ciliary epithelium and effects of pharmacological agents.

The electrical properties of the isolated monkey ciliary epithelium (CE) were determined in an Ussing-type chamber. In a Hepes, HCO3- buffered solution, transepithelial potential difference (PD), short-circuit current (SCC) and electrical resistance (Rt) were -2.5 mV (aqueous-side negative), 8.5 microA and 246 omega, respectively. Epinephrine (0.01 mM) increased the SCC and PD across the isolated monkey CE when added to the aqueous-side bathing solution but was without effect when added to the blood-side bathing solution. Forskolin (0.01 mM) increased the SCC and PD when added to the bathing solution on either side. A disulfonic stilbene, DIDS (0.1 mM), reduced the SCC and PD when added to the aqueous-side bathing solution but was without effect when added to the blood-side bathing solution. Ouabain (0.1 mM) added to the aqueous-side produced a transient stimulation followed by a gradual inhibition of the SCC and PD. On the blood-side, ouabain directly inhibited the SCC and PD towards zero. Although the general electrical properties were similar to those of the isolated rabbit iris-ciliary epithelium, there were differences in the effects of these pharmacological agents on the electrical properties.

Effects of inorganic ions on rabbit ciliary process adenylate cyclase.

The interaction of several inorganic ions (Mn2+, Ca2+, VO3-, and F-) with adenylate cyclase in the albino rabbit ciliary process particulate fraction was studied. Effects of these substances were determined on three activity states of the enzyme (basal, Gs-protein stimulated activity via isoproterenol activation of beta-adrenergic receptors, forskolin-activated catalytic unit) in the presence of 3 mM Mg2+, 1 mM EDTA and 0.2 mM EGTA. A different pattern of effects was found for each of the four ions. Addition of Mn2+ (2 mM) increased all three responses; basal by up to ten-fold, isoproterenol and forskolin by four to five-fold. Added Ca2+ (1 mM) increased basal by two to three-fold, but inhibited isoproterenol and forskolin responses by 25-50%. Added vanadate (1-10 mM) increased basal by two to four-fold, had no effect on the isoproterenol response, but doubled the forskolin response at 3 mM. Added F- (10 mM) increased basal by 30-40-fold, decreased the isoproterenol response and potentiated the forskolin response. The response to F- which directly activates G-proteins was much greater than that of non-hydrolysable GTP analogs, which also directly activate G-proteins. The results suggest that more than one type of adenylate cyclase and/or several modes of regulation of adenylate cyclase with different ionic requirements may be present in ciliary process membranes.

Modification by timolol of catecholamine stimulation of chloride transport in isolated corneas.

In the isolated frog cornea, 10(-5)M timolol completely blocked the stimulation of chloride transport by 10(-6)M isoproterenol. In this preraration, timolol inconsistently modified the response to epinephrine. In some experiments, epinephrine added to the bathing medium after timolol caused a smaller than normal increase in chloride transport, but in other instances, epinephrine caused a decrease in chloride transport. In the isolated rabbit cornea, 10(-5)M timolol totally blocked the stimulation of chloride transport by 10(-6)M epinephrine. In vivo, topical treatment of rabbit eyes with 0.5% timolol resulted in corneas from these eyes having an inhibited response to epinephrine when incubated in vitro. This inability to stimulate chloride transport persisted for several days following termination of topical treatment with timolol.

Ocular hypotension in the rabbit. Receptor mechanisms of pirbuterol and nylidrin.

Pirbuterol and nylidrin, both purported sympathomimetic amines, reduced intraocular pressure (IOP) when given topically (50 microliter, 0.1%) to albino rabbits. Pirbuterol increased the cyclic-AMP concentration in aqueous humor by a factor of 3.25, while nylidrin had no effect on aqueous cyclic-AMP nor on adenylate cyclase activity of iris-ciliary body membranes assayed in vitro. Studies of the receptor affinity of pirbuterol, timolol and nylidrin were carried out on iris-ciliary body membranes by competition binding with radioactive ligands. Four ligands were used that appear to label separate subpopulations of adrenergic receptors; dihydroalprenolol (beta-receptors), WB-4101 (alpha 1-receptors) prazosin (alpha 1-receptor subpopulation) and yohimbine (alpha 2-receptors). Pirbuterol and timolol showed exclusive selectivity for beta-receptors with high affinities (Kd 12.6 and 0.48 nM, respectively) compared with other adrenergic receptor populations in iris-ciliary body. Nylidrin had high affinities for beta-receptors (Kd 22 nM) and for the subpopulation of alpha 1-receptors labelled by prazosin (Kd 6.5 nM), but showed 100-fold lower affinity and complex binding characteristics to the two other classes of alpha-adrenergic receptor sites labelled by WB-4101 and yohimbine, respectively. The results show that pirbuterol and timolol are highly beta-receptor selective and that hypotensive responses to these drugs are not mediated by the other classes of alpha-adrenergic receptor determined in this study. However, the hypotensive response to nylidrin may be related to its prazosin-like (alpha 1-receptor) antagonist properties with additional activity at beta-receptors.

The effect of vanadate on aqueous humor dynamics in cynomolgus monkeys.

Topical administration of 1% vanadate in a formulation designed to enhance penetration lowered intraocular pressure in monkeys' eyes. The decrease in intraocular pressure was associated with significant decreases in aqueous humor flow. Tonographic outflow facility was unaltered by topical vanadate.

Effect of topically applied forskolin on aqueous humor dynamics in cynomolgus monkey.

Topical administration of a 1% forskolin suspension significantly reduced intraocular pressure in cynomolgus monkey eyes. The fall in intraocular pressure was associated with a significant (P less than 0.01) decrease in aqueous humor flow measured by a fluorophotometric technique. No significant change was found in tonographic outflow facility or pupillary diameter. A loss of effect on intraocular pressure to subsequent doses of 1% forskolin suspension occurred in cynomolgus monkeys by the third day of twice-a-day treatment.

Oxygen lowers intraocular pressure.

A significant decrease in intraocular pressure was demonstrated in 14 patients as atmospheric pressure was increased at intervals of 0.5 atmospheric pressure up to 3 atmospheres within a hyperbaric chamber. Nine of these patients had the identical protocol repeated in room air at atmospheric pressure without a significant change in intraocular pressure. Administration of 100% O2 at 15 L/min by partial rebreathing face mask to these patients using the same protocol at atmospheric pressure resulted in a significant decrease in intraocular pressure. The results in the oxygen and hyperbaric groups were not statistically different. While in the hyperbaric chamber, scleral rigidity increased uniformly, outflow facility decreased significantly, and keratometry readings remained unchanged. A significant decrease in intraocular prssure occurred in 20 rabbits that received 100% oxygen by partial rebreathing face mask for 180 minutes. Arterial blood gases were obtained at 0.90, and 180 min in seven rabbits. The pH and pCO2 did not change significantly; however, pO2 was markedly elevated. Increased oxygen concentration was felt to be responsible fot the decrease in intraocular pressure and the changes in other parameters observed in patients and rabbits.

Multiple dosing of prostaglandin F2 alpha or epinephrine on cynomolgus monkey eyes. I. Aqueous humor dynamics.

After obtaining baseline intraocular pressure (IOP) measurements for 1 week, prostaglandin (PG) F2 alpha (250 micrograms in 50 microliters saline) or epinephrine 2% solution (50 microliters) was topically applied twice daily for 2 weeks to one eye of six cynomolgus monkeys for each agent tested. Contralateral control eyes received their respective vehicles. PGF2 alpha significantly reduced IOP beginning 2 to 3 hr after the first dose, persisting thereafter. A significant (P less than 0.05) hypotensive effect remained for at least 10 hr after the first dose and at least 14 hr after the sixth dose. At 4 hr after the seventh dose, the mean reduction was 10.2 +/- 3.5 (+/- SD) mmHg below baseline (P less than 0.0025). At this time, there was also a significant (P less than 0.01) mean reduction of IOP in the contralateral vehicle-treated eyes of 6.0 +/- 3.3 (+/- SD) mmHg below baseline, which did not appear to be secondary to diurnal fluctuations, repeated tonometry, experimental manipulation, or inadvertent drug transfer. Epinephrine significantly (P less than 0.05) reduced IOP beginning 3 hr after the first dose, but this reduction was minimal and not consistent. Neither PGF2 alpha nor epinephrine altered aqueous flow as measured by fluorophotometry 2 to 6 hr after the fifth dose. Outflow facility could not be assessed by indentation tonography because IOP was often too low at the time of measurement. Whereas PGF2 alpha did not alter pupil size, epinephrine caused significant pupillary dilation.(ABSTRACT TRUNCATED AT 250 WORDS)

Multiple dosing of prostaglandin F2 alpha or epinephrine on cynomolgus monkey eyes. II. Slit-lamp biomicroscopy, aqueous humor analysis, and fluorescein angiography.

Prostaglandin (PG) F2 alpha (250 micrograms in 50 microliters saline) or epinephrine 2% solution (50 microliters) was topically applied twice daily for 2 weeks to one eye of six cynomolgus monkeys for each agent. Contralateral control eyes received their respective vehicles. A trace aqueous humor flare response occurred in some PGF2 alpha-treated eyes, which reached significance (P less than 0.05) only when observed 4 hr after the first or seventh dose. No significant anterior chamber cellular response was observed in treated as compared to control eyes. Slit-lamp biomicroscopic evaluation of the cornea, iris, and lens showed no differences in treated as compared to control eyes throughout the study. Aqueous humor samples were obtained from all eyes 4 hr after the ninth consecutive dose. Soluble protein concentration was significantly (P less than 0.01) greater in the PGF2 alpha-treated eyes (1.22 +/- 0.30 mg/ml) as compared to control (0.56 +/- 0.17 mg/ml) or to epinephrine-treated eyes (0.59 +/- 0.18 mg/ml). Microscopic examination of sediments obtained after centrifugation of the aqueous humor revealed no cells in experimental or control samples. Both PGF2 alpha and PGE2 levels were significantly (P less than 0.025) greater in PGF2 alpha-treated eyes, and showed a trend towards being greater in epinephrine-treated compared to control eyes. Neither cystoid macular edema nor other retinal abnormalities were evident by fluorescein angiography in any eyes during the second week of treatment. Multiple dosing of PGF2 alpha in monkey eyes does not appear to produce clinically significant adverse effects in either the anterior or posterior segment which would contraindicate its use in a multiple-dose clinical trial in glaucoma patients.

Flicker threshold and pattern VEP latency in ocular hypertension and glaucoma.

Latency of the pattern visual-evoked potential (PVEP) was measured in 24 ocular hypertensive (OHT) patients, eight open-angle glaucoma (OAG) patients, and 37 control subjects. The PVEP stimulus was a 2.3 cycle/degree sinusoidal grating, counterphase-modulated at 1 Hz. Field size was 9 degrees and mean luminance 1.7 log ft-lamberts. For 22 of the 32 patients, a psycholphysical measure of dynamic contrast sensitivity at 8 Hz (DRC) was obtained with a 4 degrees diameter stimulus, by determining the mean value for the contrast sensitivities to a homogeneous flickering field and to a 1.2 cycle/degree counterphase-flickering grating. Patient DRC values were compared with previously published control data from 21 subjects. Mean PVEP latencies of both the OHT and the OAG patients were greater than normal (P less than 0.001), with the OAG value larger than the OHT value (P less than 0.001). Mean DRCs were lower than normal (P less than 0.002) for both patient groups, with the OAG value lower than the OHT value (P less than 0.025). DRC correlated with PVEP latency for these patients (r = -0.66, P less than 0.001).

Vanadate effects on ocular pressure, (Na+, K+)ATPase and adenylate cyclase in rabbit eyes.

Topical administration of 50 microliter of 1% Na3VO4 caused a significant fall in intraocular pressure (IOP) in the rabbit eye at 90 min. Assay of ATPase of the iris and ciliary body in vitro at 90 min posttreatment showed no differences between control and treated (Na+, K+)ATPase, ouabain sensitive ATPase or vanadate sensitive ATPase. Accumulation of 48V-labelled orthovanadate in iris and ciliary body reached a plateau of 12 pmoles/mg dry tissue weight 4 hr after a single 25-microliter topical dose of 1% orthovanadate. In vitro inhibition of Na+ sensitive ATPase by sodium metavanadate had an IC50 of 1.8 mM, whereas a 1.8-fold stimulation of adenylate cyclase in iris-ciliary body (ICB) membranes in vitro occurred at 10 mM but not at 10 microM Na metavanadate. These results indicate that the vanadate content of the iris and ciliary body at the time of lowered intraocular pressure is too small to inhibit a significant fraction (greater than 10%) of (Na+, K+)ATPase, or cause a significant stimulation of adenylate cyclase, and that other cellular mechanisms are likely to be involved in the ocular hypotensive response to vanadate.