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Purpose: To investigate changes in ocular responses associated with the chronic administration of the adenosine A(1) agonist cyclohexyladenosine (CHA)., Methods: New Zealand White rabbits were treated unilaterally twice-a-day for 30 days with CHA (165 or 500 microg) or vehicle. Intraocular pressures (IOPs) and pupil diameters (PDs) were evaluated over the course of the study. At the end of the study period, outflow facility was determined in selected animals and compared to naïve vehicle- and CHA-treated animals., Results: In rabbits receiving 165 microg of CHA, ipsilateral IOPs at 2 and 6 hours post-drug exhibited progressively greater reduction over the course of the study. Regression analysis demonstrated a significant correlation between study duration and lower IOP at 2 and 6 hours post-drug. In rabbits receiving 500 microg of CHA, ipsilateral IOP reductions at 2 hours post-drug were similar throughout the 30-day study. However, analysis of ipsilateral IOPs 6 hours following CHA administration, demonstrated a significant correlation between study duration and lower IOPs. Enhanced contralateral responses at 2 hours post- drug, were also measured in rabbits receiving 165 or 500 microg of CHA. In animals receiving chronic CHA treatment for 30 days, outflow facility 3 hours post-CHA was significantly elevated over that measured in naïve vehicle-treated rabbits. Although mean outflow facility in chronic treatment animals was slightly elevated over CHA-induced increases in naïve rabbits, this difference was not significant. No evidence of tolerance was observed for either dose during the course of these studies. No change in PD during the course of these studies was measured., Conclusions: The chronic administration of the adenosine A(1) agonist CHA twice daily produced no evidence of tolerance. Unexpectedly, the IOP response to CHA was enhanced with chronic administration. These data provide evidence that the use of adenosine A(1) agonists may be useful in the chronic treatment of ocular hypertension at doses lower than those identified in acute IOP studies.

Purpose: To evaluate the effects of a novel daunomycin (DM) implant on intraocular pressure (IOP), bleb survival, and anterior segment complications when administered during filtering surgery on New Zealand White rabbits., Methods: Implants were prepared by covalent coupling of DM to a high molecular weight hyaluronic acid (HA) and fabricated into solid implants containing 250, 65, and 25 microg of DM. Full thickness sclerostomies were performed, and rabbits received no implant (control), placebo implant containing HA, or an implant containing HA-DM conjugate at the time of surgery. Rabbits were then followed for 30 days to assess change in IOP, bleb survival, and anterior segment associated complications., Results: In vitro, the release of DM from the implants was a first order process with a half-life of 51 h. In control rabbits, rabbits receiving placebo implant, and rabbits receiving HADM (250 microg) implants, the mean IOPs on day 3 were 11.1 +/-1.6, 10.8 +/- 2.7, and 14 +/- 0.98 mm of Hg, respectively. On days 5 through 9, IOP in the control and placebo-implanted groups returned to preoperative levels. However, in rabbits receiving 250 microg of conjugated DM, mean IOP on day 7 was reduced from preoperative levels by 11.8 +/- 3.2 mm of Hg (P < 0.05). This reduction in IOP was significantly different (P < 0.05) from both control and placebo implant groups, and IOPs remained at these levels until studies were terminated on day 30. In control and placebo-implanted rabbits, bleb size started decreasing on day 1, and by day 7, no blebs were observed. In rabbits receiving 250 microg HA-DM implants, mean bleb survival time was greater than 30 days. The DM-induced reduction in IOP and enhanced bleb survival was dose-dependent. In rabbits receiving 65 microg of conjugated DM, lOPs were significantly reduced through day 30; however, at times beyond day 19 there was a gradual rise in mean IOP as filtering procedures in individual animals began to fail. Mean bleb survival time was greater than 30 days for implants containing 65 microg of conjugated DM. In rabbits receiving HA-DM implant containing 25 microg of DM, IOPs were not significantly different from preoperative levels beyond day 9, and no significant enhancement in bleb survival was observed in these animals. Comparison of HA-DM conjugated implants to those containing equal doses of free DM demonstrated that the mean IOP change at 30 days were similar; however, there was a reduction in anterior segment complications associated with the use of HA-DM conjugated implants., Conclusions: This study provides evidence that the controlled release of DM when conjugated to HA can significantly improve the success of filtering procedures. The maintenance of ocular hypotension and bleb survival along with the reduction in anterior segment complications supports the idea that HA-DM conjugate will be more efficacious than the use of free DM in improving the success rate of filtering surgery.

Purpose: To characterize the adenosine receptor modulation of norepinephrine (NE) release from sympathetic neurons in the isolated rabbit iris/ciliary body., Methods: Iris/ciliary bodies were isolated from New Zealand White rabbits and incubated in the presence of 3H-NE. Norepinephrine release was elicited by field stimulation with varied frequencies from 5 to 30 Hz. The effects of adenosinergic and alpha 2 adrenergic compounds on NE release were determined and compared., Results: At a stimulation frequency of 5 Hz, the addition of the adenosine A1 agonist CHA did not significantly alter 3H-NE release. However, in the presence of the alpha 2 adrenergic antagonist yohimbine, the addition of CHA produced a dose-related reduction in 3H-NE release. The EC50 for this reduction was 14 nM. At a stimulus frequency of 20 Hz, the addition of CHA alone (10(-6) M) produced a significant reduction in 3H-NE release of 41%. The EC50s for the adenosine A1 agonists CHA- and R-PIA-induced suppression of 3H-NE release at 20 Hz were 32 and 24 nM, respectively. The adenosine A2 agonist CV-1808 did not alter 3H-NE release at stimulation frequencies of 5 or 20 Hz. Pretreatment of tissues with the adenosine A1 antagonist CPT or pertussis toxin reversed the suppression of 3H-NE release induced by CHA. Comparison of the inhibitory responses of CHA to the alpha 2 adrenergic agonist UK-14,304 at stimulus frequencies of 5 to 30 Hz demonstrated that this adenosine A1 agonist was effective only in suppressing NE release at frequencies of 20 Hz or greater. In contrast, the alpha 2 adrenergic agonist UK-14,304 was most effective in reducing NE release at 5 Hz., Conclusions: These results provide evidence that adenosine agonists inhibit 3H-NE release in the iris/ciliary body via prejunctional adenosine A1 receptors linked to Gi/o-protein. However, the expression of this response was dependent on the frequency of neuronal stimulation. Hence, prejunctional adenosine A1 receptors may act to selectively limit high-frequency neurotransmission.

Cyclodextrins have been used to improve drug solubility, stability and absorption for oral and parenteral administration. However, their potential for improving ocular drug delivery has received little attention. To evaluate the ability of hydroxypropyl-beta-cyclodextrins to improve ophthalmic drug bioavailability following topical administration, the miotic effect of topical solutions of pilocarpine was studied in New Zealand White rabbits. Pilocarpine varying in dose from 5 to 500 micrograms in the presence or absence of 5% cyclodextrin was administered (50 microliters) topically and the change in pupil diameter determined. These results demonstrated that pilocarpine alone or in the presence of cyclodextrin produces a dose-related reduction in pupil diameter. The addition of cyclodextrins produced a significant left-shift in the dose response curve, with an ED50 of 64 micrograms and 19 micrograms for pilocarpine and pilocarpine/5% cyclodextrin solutions, respectively. Studies in which the concentration of cyclodextrin was varied revealed that a one-to-one molar ratio of pilocarpine to cyclodextrin was sufficient to provide maximum increase in pilocarpine bioavailability. Electrophysiology and scanning electron microscopic studies demonstrated that cyclodextrin does not disrupt the normal ion transport currents, barrier properties or surface features of the corneal epithelium. Viscosity measurements indicated that difference in the viscosity of pilocarpine and pilocarpine/cyclodextrin solutions cannot account for increased bioavailability of pilocarpine. These data support the idea that the addition of cyclodextrin significantly improves the ocular bioavailability of pilocarpine. This enhanced bioavailability of pilocarpine does not appear to be due to a mechanism destructive to the epithelium or to an increase in vehicle viscosity.

Previous studies have shown that ocular alpha 2 adrenoceptors are located prejunctionally on sympathetic neurons and postjunctionally on cells in the iris/ciliary body. While the activation of alpha 2 adrenoceptors at each site has been postulated to alter aqueous humor dynamics, little is known about the pharmacological characteristics of these receptors or their role in the modulation of anterior segment function. The purpose of the current study was to determine the possible heterogeneity of ocular alpha 2 adrenoceptors using relatively selective alpha 2 adrenoceptor agonists and antagonists to examine ocular pre- and postjunctional alpha 2 adrenoceptors. Prejunctional alpha 2 effects were evaluated by means of the cat nictitating membrane (CNM) preparation. Postjunctional alpha 2 effects were evaluated by means of the cAMP assay in rabbit iris root/ciliary body. In the CNM, the administration of UK-14, 304 (UK) produced a dose-related inhibition of neuronally mediated contractions. Pretreatment with the alpha 2 antagonist rauwolscine caused a 1 to 2 log unit right shift in the dose-response curve of UK in the CNM. However, pretreatment with alpha 2 antagonist SKF 104078 had no demonstrable effect on UK-induced inhibition of neuronally mediated contractions of the CNM. In the rabbit iris root/ciliary body, UK produced a concentration-dependent inhibition of cAMP accumulation on isoproterenol- and VIP-induced cAMP production. Pretreatment of iris root/ciliary bodies with SKF 104078 or rauwolscine reversed the inhibitory effect of UK on isoproterenol- and VIP-induced accumulation of cAMP. These data provide the first evidence that the pre- and postjunctional alpha 2 adrenoceptors represent pharmacologically distinct subpopulations of receptors in the eye.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine A1 agonists have been shown to induce a variety of pharmacological effects. In New Zealand White rabbits, the topical administration of 500 micrograms of the relatively selective adenosine A1 receptor agonist R(-) phenylisopropyladenosine (R-PIA) produced a biphasic response in IOP in the ipsilateral eye: an initial ocular hypertension (3.5 +/- 1.4 mm of Hg) at 0.5 hour, followed by significant reduction in IOP (5 to 8 mm of Hg) from 2 to 6 hours postadministration. The IOP response to 50 and 165 micrograms of R-PIA demonstrated that the ocular hypotensive response to R-PIA was dose-related; however, no initial hypertension was observed at these lower doses. The ocular response to R-PIA was primarily unilateral with only a small reduction in contralateral IOP at 1 hour observed in animals treated with 500 micrograms. No significant change in pupil diameter was observed with any dose of R-PIA. Pretreatment with the adenosine antagonist CPT (10 mg/kg; i.p.) significantly inhibited the ocular hypotensive response to R-PIA. However, pretreatment with the cyclooxygenase inhibitor indomethacin (50 mg/kg; i.p.) did not alter the change in IOP induced by R-PIA. The administration of R-PIA once a day for five days demonstrated that tolerance does not develop in rabbits with repeated administration. These data demonstrate that the adenosine A1 agonist R-PIA can lower IOP. The unilateral nature and the inhibition by CPT supports the idea that this response is mediated by adenosine receptors located in the eye.(ABSTRACT TRUNCATED AT 250 WORDS)

The nervous system has profound modulatory influences on many inflammatory processes, particularly within the eye. These properties are in part mediated via neuropeptides. The neurotoxin, capsaicin, has been utilized as a valuable experimental tool to study the role of neuropeptides in many organ systems. Retrobulbar injection of capsaicin into rats results in the loss of sensory nerve function with rapid onset of inflammation (6-12 hours) which is confined to the the anterior segment. The hallmark of the acute response at 24-48 hours is marked polymorphonuclear leukocyte (PMN)/mononuclear cell influx and opacification of the corneal stroma with degeneration and loss of the central epithelium. PMN/mononuclear cell infiltration was also evident within the angle, the anterior chamber, and the iris. The corresponding posterior segments were normal. There was extensive corneal neovascularization between 7 and 14 days. This keratouveitogenic response in rats was age-dependent and can be attenuated by prior systemic pretreatments with capsaicin. This model should prove to be useful in the study of mechanisms of intraocular neurogenic inflammation.

Tumor-promoting phorbol esters have been shown to modulate a number of physiological events in various cell types, and these events are associated with the activation of protein kinase C. The purpose of this study was to examine the effects of phorbol ester stimulation on protein kinase C activity and epithelial ion transport in the Cl- secreting rabbit corneal epithelium. We report here that nanomolar concentrations of tumor-promoting phorbol ester, 12-0-tetradecanoyl-phorbol-13 acetate (TPA), stimulate active ion transport via a putative receptor located in the corneal epithelium. This stimulation is a Cl- -dependent process, but is independent of beta-adrenergic receptor activation and prostaglandin formation. Biochemical data support the idea that TPA activates Ca++/phospholipid-dependent protein kinase C in the corneal epithelium. Therefore, we suggest that protein kinase C may have a role in the regulation of membrane Cl- transport in the mammalian corneal epithelium.

The selective DA2 agonist, N-0437, produced an acute reduction in intraocular pressure (IOP) and pupil diameter (PD) when applied topically to the eyes of normal monkeys. Ocular hypotension and miosis were primarily unilateral in nature, dose-related and lasted 3 to 5 hours following drug instillation. In normal monkeys, 24 to 48 hours following drug administration, a secondary chronic (greater than 24 hrs) reduction in pressure was observed. Once-a-day topical administration of N-0437 (250 micrograms), to normal monkeys, for 4 days produced a chronic unilateral reduction in IOP that persisted for 18 days. Associated with this reduction in pressure on day 2 through 6 were miosis and ptosis of the treated eye. Although topical administration did not lower IOP in rabbits, intracameral injection of N-0437 significantly depressed IOP for 3 days when compared to control injected eyes. Evaluation of ocular sympathetic innervation in N-0437 treated rabbits indicated that these fibers were not functional. In rabbits, intracameral administration of the active (S,-) and inactive (R,+) enantiomer of N-0437 produced equivalent reductions in pressure. These data provide further support for the hypothesis that DA2 receptor agonists can produce acute reductions in IOP. In addition, N-0437 appears to have a second non-receptor mediated mechanism of action that produces a chronic reduction in IOP. This chronic reduction in pressure appears, in part, to result from an interruption of ocular sympathetic nerves function.

The corneal epithelium of the rabbit is functionally characterized as a "tight" ion transporting cell layer. Its barrier properties arise from the high electrical resistance of both the outermost cell membranes and the paracellular zonulae occludens. The specific ion transport process and secondary fluid transport by this tissue are modulated via cyclic AMP. There is evidence for the autonomic control of C1- secretion by the corneal epithelium.