Afrial Natriuretic Peptides - IOVS

Investigative Ophthalmology & Visual Science, Vol. 30, No. 11, November 1989 Copyright © Association for Research in Vision and Ophthalmology

Afrial Natriuretic Peptides Effects on Intraocular Pressure, cGMP, and Aqueous Flow Michael S. Korenfeld and Bernard Decker We report a significant decrease in intraocular pressure after intravitreal injection of atrial natriuretic peptide (AP). There is also an increase in cGMP in iris-ciliary body preparations after exposure to physiologic concentrations of AP in vitro. We present evidence that after intravitreal AP administration there is a decrease in aqueous humor flow associated with the decrease in intraocular pressure. The changes in aqueous humor flow were estimated by automated fluorophotometry and aqueous humor ascorbate concentrations. Invest Ophthalmol Vis Sci 30:2385-2392,1989

and an increase in guanylate cyclase activity21'23'24 result when ciliary processes are exposed to physiologic concentrations of AP. There are no data addressing changes in the rate of aqueous formation in response to APs, and this question was explored in this study.

The atrial natriuretic peptides (APs) are synthesized, stored, and released by cardiac atrial myocytes.1'2 Extraatrial sites of AP synthesis have been described34; however, no mRNA transcripts of APs have been localized in ocular tissue.5 These peptides have direct natriuretic, diuretic, and vasodilatory properties.6'7 They also interact with many hormonal systems,8"" most of which are directly or indirectly involved in fluid and electrolyte homeostasis. Additionally, APs bind to the choroid plexus, resulting in a decrease in cerebrospinal fluid production.12 In the eye, APs bind to specific receptors. The principal receptor for APs is the membrane-bound particulate guanylate cyclase molecule,1314 although recent evidence suggests that AP receptors are more heterogeneous than previously thought.15"17 There is a very high density of receptors in the ciliary epithelium, in which they have been identified by both light microscopic18"20 and electron microscopic20 autoradiography. There is a lower density of specific receptors localized to the choroid, choriocapillaris, ciliary musculature, and retina. There are several reports of a reversible decrease in intraocular pressure after intravitreal,21"24 intravenous,25 and intracameral22 AP administration. There is also evidence that a decrease in basal and stimulated adenylate cyclase activity20

Materials and Methods All atrial peptide analogs utilized (API, II, and III) were generously supplied by the Department of Pharmacology of Washington University (St. Louis, MO) and by Monsanto/Searle (St. Louis, MO) and were between 82% and 95% free base of the designated compound. Lyophilized APs were aliquoted at 1.0 mg/ml in sterile 0.9% saline with 0.5% BSA and frozen at -70°C in siliconized glass tubes until needed. Aliquots were thawed on ice, and the required dilutions were made in the aliquot tube. Diluted AP solutions were stored on ice during experiments. Except for the APs, all chemicals were obtained from Sigma Chemical Co. (St. Louis, MO). Animals

New Zealand albino rabbits, 1.8-3.5 kg, were housed and handled according to the ARVO Resolution on the Use of Animals in Research. Rabbits were entrained with 12-hr light-dark cycles for at least two weeks prior to the experiments. Prior to baseline intraocular pressure measurements, conscious animals were wrapped in restraining cloths and allowed to equilibrate in their bound state. All animals receiving injections into the globe were premedicated with 50 mg/kg intraperitoneal indomethacin just prior to being restrained in wrappers. All animals were euthanized by anesthetization with 5 mg/kg xylazine intramuscularly and 60 mg/kg ketamine intramuscularly followed by intracardiac air embolization.

From the Department of Ophthalmology, Washington University School of Medicine, St. Louis, Missouri. Supported in part by Teaching Grant 5 T32 EY-07057-07 from the National Eye Institute, Bethesda, Maryland, and a Glaucoma Research Grant from The American Health Assistance Foundation, Rockville, Maryland. Submitted for publication: October 27, 1988; accepted May 8, 1989. Reprint requests: Michael S. Korenfeld, MD, Department of Ophthalmology, University of Missouri, Columbia, One Hospital Drive, Columbia, MO 65212.

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Intraocular Pressure Measurement

All intraocular pressure (IOP) measurements in conscious animals were made with an Alcon Pneumotonometer after topical anesthesia with 0.5% proparacaine. Pneumotonometer IOPs were referenced periodically with manometric IOPs to ensure accuracy. Manometric IOP measurements were made with manometric intracameral needles in systemically anesthetized animals and in conscious, topically anesthetized animals who had awakened from systemic anesthesia. The sharp ends of the 23-gauge intracameral needles were occluded with cyanoacrylic glue, a side port was drilled to their lumens, and the hub ends were broken off and attached to PE-50 tubing (Clay Adams). Cannulation was achieved by placing the needle across the anterior chamber and entering and exiting at the limbus. A continuous fluid column passed from the calibrating water column, over the pressure transducer, through the tubing, and into the fluid of the anterior chamber. The transducer was precalibrated each day on a Sanborn recorder. Anesthesia for manometric IOP experiments was achieved either with 5 mg/kg xylazine intramuscularly, followed five min later with 60 mg/kg ketamine intramuscularly, or with 1.0 g/kg urethane intravenously. Urethane was chosen to achieve long-lasting, stable anesthesia. Once the animals were anesthetized, femoral arterial cannulas were placed under sterile conditions and under infiltrating anesthesia with 0.5% marcaine. The arterial pressure line was connected to a precalibrated Sanborn pressure transducer. Recordings of the IOP and systemic arterial blood pressure were made continuously and simultaneously. Drug delivery was accomplished after baseline values for these parameters were established. Intracameral experiments: Two different side-port needles were placed through the cornea, as described above, with their side ports facing away from each other. The tubing leading from these needles was attached to a push-pull device constructed from a matched pair of 50-/A Hamilton syringes with a common plunger. The two sides of these lines were preloaded either with 0.1 mg/ml APIII containing dilute fluorescein or with vehicle. With this instrument, the drug solution was delivered while the same volume of aqueous humor was removed from the other port to prevent any acute IOP changes. Up to 50 ii\ of drug solution was injected at a time with no more than 2 mmHg acute IOP change. The fluorescein tracer ensured drug delivery and minimal loss of drug into the receptacle port. IOP was measured manometrically. Intravenous experiments: Conscious, topically anesthetized animals or animals with manometric IOP needles and arterial cannulas were used for intra-

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venous drug delivery. Intravenous access was established in the marginal ear vein after infiltration with 0.5% marcaine. With an infusion pump (Razel Industries), APIII or vehicle was delivered intravenously on a continuous basis. Delivery rate of APIII was 0.1, 0.3, 1.0, 3.0, and 10.0 ^g/kg/min for at least 30 min. Intravitreal experiments: Restrained conscious animals had 10-/il injections of 0.2 jig/ml to 1.0 mg/ml APIII in 0.9% saline with 0.5% BSA or vehicle into the midvitreous body. Injections were performed by inserting a 27-gauge needle on a Hamilton syringe through the superior rectus muscle26 to prevent leakage. Subconjunctival experiments: Subconjunctival injections were made with 100 ix\ of a 0.2 mg/ml solution of APIII in 0.9% saline, 0.5% BSA solution or this same vehicle diluted 1:1 with dimethylsulfoxide (DMSO). A 27-gauge needle was introduced at the limbus after topical anesthesia with 0.5% proparacaine. Topical experiments: Solutions of API, II, and III were dissolved in several vehicles at concentrations ranging from 0.003 to 1.0 mg/ml. Vehicles included 0.9% saline, 0.5% hydroxymethylpropylcellulose (Allergan), 0.5% hydroxymethylpropylcellulose:DMSO 1:1, and 0.9% saline:DMSO 1:1. Fifty microliters of these solutions were delivered to the superior limbus and observed to cover the cornea. The eyes were held open for approximately 10 sec after drop applications. Drops were administered at time 0 and 15, 30, and 60 min later. cGMP Determinations Rabbits were euthanized as previously described, and iris-ciliary bodies were dissected, quartered, and immediately placed in 5°C oxygenated Krebs' solution. The tissue was allowed to equilibrate in this medium for at least 30 min. The tissue was then transferred to a test tube containing 37°C oxygenated Krebs and was maintained at this temperature and continuously oxygenated for 15 min (preincubation). One quarter of each iris-ciliary body was then placed into 37°C solutions containing (1) Krebs' alone; (2) 1-mM isobutylmethylxanthine (IBMX, a phosphodiesterase inhibitor) in Krebs'; (3) 10-nM APIII in Krebs'27; or (4) 1-mM IBMX and 10-nM APIII in Krebs'. The four quarters of a single ciliary body were kept together during the four different incubations and subsequent processing. Incubations were performed for 0.5, 1, 5, and 10 min. At the end of the incubation the tissue was immediately placed into cold 10% trichloroacetic acid and homogenized in ground glass conical tubes. The tissue was then prepared for the cGMP assay as previously described.28

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ATPJAL NATRIURETIC PEPTIDE5 / Korenfeld and Decker

Samples were frozen at -70°C until the cGMP assay was performed. cGMP assay: All samples were thawed on ice the day of the assay. Aliquots of the samples were acetylated with an acetic anhydride:triethylamine (1:2) solution. The cGMP radioimmunoassay and antibodies were identical to those used elsewhere.28 Aqueous Humor Dynamics

Automatedfluorophotometry: Approximately 14 hr prior to an experiment, the rabbits' corneas were loaded topically withfluorescein.One drop of Fluress (0.25% fluorescein and 0.4% benoxinate; Barnes Hind, Sunnyvale, CA) was placed in both eyes every 5 min for 1 hr. After this loading period, the eyelashes and whiskers were trimmed and the lids were washed briefly to remove hair and excessfluoresceinfrom the fur. The next morning baseline measurements of corneal and anterior chamber fluorescein concentration were obtained using a Fluorotron Automated Fluorophotometer. Measurements at each time point represented a time- and fluorescein-concentration average of at least three samplings of a single eye. Three baseline measurements were obtained, separated by at least 30 min. A pediatric lid speculum was used to expose the cornea during measurements. One drop of balanced salt solution (BSS; Allergan, Irvine, CA) was placed on the cornea as needed to provide an optically smooth corneal-air interface. Baseline aqueous flow rate and transfer coefficients were obtained, and calculations were performed to ensure that these parameters were based on first-order kinetics for fluorescein disappearance. After first-order kinetics were verified, rabbits received intravitreal injections of 10-jug APIII or vehicle, as previously described. Approximately 1 hr after intravitreal injection, scanning was begun in triplicate for each eye. Scans were performed alternating between eyes until the fluorescein signal became too inaccurate to measure or until the precision of the triplicate measurements became unreproducible. In order to calculate the effect of the 10-/xg APIII intravitreal injection on aqueous formation, data obtained close in time were averaged with a modified Yablonski program.29 Since more than one animal was used per day and since eyes were tested in succession, post-injection data were collected at different times post-injection in different animals. For statistical analysis, however, two eyes were treated as though data were obtained simultaneously, although a pair of measurements from the left and right eyes were actually obtained over 8-15 min. The measurements of Ko (the fraction of existing aqueous which leaves the anterior chamber per unit time) of the control eye were subtracted from the Ko of the experimental eye and plotted against time after intravitreal injection.

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Each eye was referenced against its own baseline. In order to average the effects for all animals, interpolations of each animal's tracing were made to generate values for test - test baseline] [control - control baseline]\ test baseline J [ control baseline J/ at 20-min intervals between the initial and final readings for that animal. This handling of the data did not require the anterior chamber volume for calculation of relative aqueous flow. Statistics were done for each time interval in which N > 10. An N < 10 was deemed too small for statistical evaluation. Ascorbate method: Restrained conscious animals had intravitreal APIII or vehicle injections, as previously described.26 IOP determinations were made each hour for 4 hr after injection. Four hours after intravitreal injection, posterior and anterior chamber aqueous humor were collected and immediately titrated using the metaphosphoric acid method.30 Results were calculated with the formula described previously.30 All statistical analyses in this study compare changes in each eye to its own baseline and then to each other when possible. This was done to account for diurnal variations in intraocular pressure31 as well as the inherent variability of baseline measurements. A two-tailed paired student t-test was used to compare the test and control eyes. Aqueous humor AP concentration: Two groups of animals were studied. One group had 10-/ul intravitreal injections of either 10 ng APIII or vehicle at time 0 as described previously. Sixty minutes later, 70-/ul anterior chamber aqueous humor samples were obtained from restrained animals anesthetized topically with 0.5% proparacaine. A 27-gauge needle fitted on a Hamilton syringe was directed through the limbus toward the posterior central cornea. Samples were immediately frozen on dry ice and AP immunoreactivity was assayed as described elsewhere.32 A second group of animals had a baseline paracentesis in one eye followed by a 1.0 /ig/kg/min intravenous infusion of APIII for 20 min. Just after the infusion, a 70-/xl aqueous humor sample was obtained from the other eye. Results Intraocular Pressure

After intracameral administration of AP, no significant changes in IOP were observed. We therefore could not confirm the IOP decrease reported elsewhere22 after intracameral AP administration. In animals receiving continuous intravenous infusions of AP there was a definite decrease in IOP in

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Table 1. Dose and duration of APIII infusion

Time of maximal change of IOP Maximal change of IOP from baseline (mmHg ± SEM) Change of MAP at the time of maximal IOP change (mmHg ± SEM) Time to return of baseline IOP

Re-achieved baseline IOP compared to initial IOP Mean arterial pressure at the time of re-achieved baseline IOP

1.0 fig/kg/min X 15 min

3.0 fig/kg/min X 75 min

10 min into infusion

20 min post-infusion

-1.1 ±0.4, N = 6

0.3 iig/kg/min X2hr

1.0ng/kg/min X 30 min

end of infusion

end of infusion

-2.5 ± 0.5, N = 6

+0.2 ± 0.6, N = 6

-0.4 ± 0.2, N = 6

-12.2 ±3.2, N = 6

-21.7 ± 3.0, N = 5

+6.7 ± 6.4, N = 3

-5.3 ±0.7, N = 4



—


-0.1 ±0.1,N = 6

-0.7 ± 0.6, N = 6

—

-0.4 ± 0.2, N = 6

0.0 ± 0.8, N = 6

-0.5 ± 2.4, N = 5

—

+0.3 ± 1.2, N = 4

response to the drug; however, there was a concomitant decrease in systemic blood pressure. In general, for every 10 mmHg decrease in the mean arterial pressure (MAP), there was approximately 1 mmHg decrease in IOP. Two animals experienced small, reversible IOP decreases with a steady or increased blood pressure, but in general, IOP and MAP varied directly (Table 1). It was hoped that a longer infusion of APIII (0.3 /wg/kg/min for 2 hr) would allow the IOP changes to be observed without systemic blood pressure effects, but this did not occur. After intravitreal injection of 0.002-1.0 ^g APIII, no significant IOP change was observed. After a 10-^g injection, however, a statistically significant IOP decrease was first seen at 1 hr and was maximal at 4 hr (Fig. 1). There were no statistically significant IOP changes after topical or subconjunctival administration of any AP analog in a wide range of concentrations and in different vehicles. cGMP In vitro assays of iris-ciliary body tissue in the presence of 1 mM IBMX or 10-nM APIII and 1 raM IBMX together showed a statistically significant increase in tissue levels of cGMP over medium alone (Fig. 2). However, the increase in cGMP over baseline in tissue exposed to 10 nM APIII alone was not statistically significant. There was a statistically greater accumulation of cGMP in tissue exposed to APIII plus IBMX when compared to either APIII or IBMX alone in the 5- and 10-min incubations. There was also a significant increase in cGMP at 1 min when AP plus IBMX was compared to IBMX alone. Aqueous Flow

Fluorophotometry: Statistically significant decreases in aqueous formation were observed in eyes

receiving intravitreal APIII during the period of 140-220 min after injection. The magnitude of the decrease in aqueous formation during this period was approximately 20% (Fig. 3). Ascorbate method: Four hours after 10 ^g APIII was delivered intravitreally to one eye, there was no statistical difference between the two eyes in anterior chamber aqueous humor ascorbate concentrations. In the posterior chamber aqueous humor however, there was a significant increase in ascorbate concentration in the test eye as compared to the control. This increase in the posterior chamber ascorbate

p< 0.001

1

2

3

4

TIME AFTER INJECTION (hours)

Fig. 1. Change in intraocular pressure, (test value - test baseline) - (control value - control baseline), after intravitreal injection of 10 ^g APIII. Mean ± SEM, N = 25 except at 4 hr post-injection when N = 22. Two-tailed paired student t-test.

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40 30

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20

JB

Interval of significant decrease of K

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0.2000

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0 0.51.0

5.0 TIME OF INCUBATION (minutej)

Fig. 2. Change in concentration of cGMP in iris-ciliary bodies in vitro. ( • — • , Krebs' medium; • — • , 10 nM APIII in Krebs'; A —A, 1 mM IBMX in Krebs'; A—A, 10 nM APIII + 1 mM IBMX) Mean ± SEM, N = 3. Two-tailed paired student t-test. 1 min: IBMX vs Control P < 0.05 AP + IBMX vs Control P < 0.02 AP + IBMX vs IBMX P < 0.05 5 min: IBMX vs Control P < 0.01 AP + IBMX vs Control P < 0.01 AP *+ IBMX vs AP P < 0.02 AP + IBMX vs IBMX P < 0.01

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