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Immunoreactive atrial natriuretic factor in aqueous humor: Its concentration is increased with high intraocular pressure in rabbit eyes
b’~~tonRes. Vol. 30, No. 9. pp. 1305-1310, 1990 Printed in Greal Britarn. All rights reserved
Copyright
~2-69~9/90 $3.00 + 0.00 Q 1990 Pergamon Press pit
IMMUNOREACTIVE ATRIAL NATRIURETIC FACTOR IN AQUEOUS HUMOR: ITS CONCENTRATION IS INCREASED WITH HIGH INTRAOCULAR PRESSURE IN RABBIT EYES RAQUEL FER~A~DEZ-DURAN~,’ ALBERTOTRIVIIQO,’JOSEM. RAMIREZ,* MARIO GARCIA DE LACOBA,’ ANA RAMIRE&* JUAN J. SALAZAR,’ ARTURO FBBNANDEZ-CRUZ’ and JOLANTAGUTKOWSKA’ ‘Diabetic, Hypertension and Obesity Unit and 2Department of Ophthalmology, Laboratory of Ocular Morphology, Hospital Universitario San Carlos, Ciudad Universitaria, 28040-Madrid, Spain and 31nstitut de Recherches Cliniques de Montreal, I10 Avenue des Pins Ouest, Montreal, Canada H2W 1R7 (Received 9 February 1989; in revisedform
25 October 1989)
Abstract-Atria1 natriuretic factor (ANF) concentration in the aqueous humor (AH) was studied in rabbits with experimental glaucoma induced by injecting a-chymotrypsin into the posterior chamber. In normal rabbit eyes, the ANF concentration in AH was 3.1 f 1.2 pg/ml (% & SEM; n = t2), ranging from 0 to 5.8 pg/ml, whereas it was si~i~cantly higher in AH from glaucomatous rabbit eyes, being 8 1.O+ 9.8 pg/ml (n = 12). These findings were correlated with intraocular pressure (IOP), which was 13.0 + 2.4 mmHg (n = 12) in normal rabbit eyes and significantly greater in glaucomatous eyes: 24.4 f 3.0 mmHg (n = 12). Our data indicate that enhanced ANF release in AH during experimental glaucoma may play an important physiological role in modulating IOP. Aqueous humor
Atria1 natriuretic factor
Experimental glaucoma
INTRODUCTION
A great deal of evidence suggests that atria1 natriuretic factor (ANF) modulates body volume-salt homeostasis (de Bold & Flynn, 1983). Originally found in mammalian atria, ANF is not exclusively an atria1 hormone. Its presence has been demonstrated in many other tissues, such as heart ventricles, lung, various parts of the brain, anterior uvea and retina as well as blood and ascitic fluid. Immunoreactive ANF concentrations in the anterior uvea and retina of the rat are 31 and 8 rig/g tissue, respectively (Stone & Glembotski, 1986). A high density of ANF receptors has been observed on the epithelial side of the ciliary body (Quirion, Dalpe, DeLean, Gutkowska, Cantin & Genest, 1984; Bianchi, Anand-Srivastava, de Lean, Gutkowska, Forthomme, Genes1 & Cantin, 1986). Guanylate cyclase, the proposed transducer of the ANF signal to the intracellular compartment, is stimulated by ANF in membranes derived from ciliary processes (Mittag, Tot-may, Ortega & Severin, 1987). Furthermore, ANF has been shown to decrease intraocular pressure (IOP) in numerous species (Sugrue & Viader, 1986; Nathanson, Hunnicutt
Intraocular pressure
& Owen, 1987). Since IOP is increased in glaucoma, we decided to study the probable presence of ANF in ~aucomatous eyes to evaiuate the hypothesis that it may modulate IOP. MATERlALS
Experimental
AND METHODS
glaucoma
Adult, male, New Zealand albino rabbits (3.0-3.5 kg) were maintained under alternating 12-hr periods of darkness and light on laboratory chow and water ad libitum. Experimental glaucoma was produced in the right eye by injecting 0.86mg of ~-chymotrypsin diluted in 0.13 ml of saline into the posterior chamber according to a modification (Ramirez, Triviiio & Garcia, 1986) of the method of Vareilles, Durand, Siou and Le Douarec (1987). The left eye was injected with saline. IOP was measured bilaterally by aplanation tonometry. A local anesthetic (0.2% fluorescein-0.4% benoxinate) was given topically, and the tonometer (Draeger, Moller Wedel, Germany) was applied tangentially to the cornea for 5 sec. 40 days after the a-chymotrypsin injection, the animals were anesthetized by intravascular administration of
1305
RAQLIELFERNANDEZ-DWWW et ai
1306
sodium pentobarbital (30 mg/kg) and AH was drawn with a 1.0 ml-syringe. The needle (dia. 0.3 mm) was forced through the cornea1 scleral junction into the anterior chamber. The rabbits were then killed by anesthetic overdose and their eyes were immediately enucleated for dissection, which was followed by embedding in paraplast (Sherwood Medical, Ireland) and staining with hematoxylin-eosin. Measuremenr
of ANF
The aqueous humor (AH) samples were collected in chilled tubes containing protease inhibitors at the following final concentrations: 1 mg~m1 EDTA, 10e3 M phenylmethylsulfonyl fluoride (PMSF) (Sigma No. P-7626) and 5 x low6 M pepstatin A (Sigma No. P-4265). ANF extraction was performed according to the method used for plasma ANF (Gutkowska, Bonan, Roy, Bourassa, Garcia, Thibault, Genest & Cantin, 1986). Briefly, the Sep-Pak C-l 8 cartridges (Waters Associates, Milford, MA) were activated by washing with 8-10 ml acetonitrile then washed with 8-10ml ammonium acetate (0.2% at pH 4.0). The AH samples were then applied on the cartridges, washed with 5 ml ammonium acetate (0.2%, pH 4.0) and the absorbed ANF was eluted with 3 ml acetonitrile (60%) in ammonium acetate (0.2%, pH 4.0). The organic solvent was evaporated under nitrogen stream followed by lyophiliration. The residue was taken up in RIA buffer (0.1 M phosphate, pH 7.4) containing 0.3% NaCl, 0.1% BSA and 0.1% Triton X- 100. Immunoreactive ANF was measured by a specific and sensitive radioimmunoassay (RIA), as previously described (Gutkowska, 1987). The detection limit was 0.75 pg with a within and between assay variability of 10.2 and 18.7%, respectively. The recovery of standard ANF (34.5 and 69pg/ml) added to pooled AH was 79.0 + 3.2% (n = 10). Data analysis
The statistical significance of the differences between groups was evaluated by Student’s f-test. RESULTS
IOP was increased in the eyes of 12 out of 16 rabbits as soon as 72 hr after the cc-chymotrypsin administration. After 40 days, it was significantly higher than in rabbit eyes injected with saline alone (Table I).
Table
1, lmmunoreactive ANF in AH and IOP d norma: and glaucomatous rabbit eyes ANF
Normal eyes (n = 12) Glaucomatous eyes (n =
@gimf)
3.1 f I.2
12) 8I .? + 9.8’
10P (mmHg)
13.0& 2.4 24.4f 3.0,
Values are given as means ,+ SEM statistically different from normal values, *P c. 0.05.
Mo~hologi~ally, eyes treated with the enzyme had a buphthalmic appearance with tears in the Descemet’s membrane and a discrete cornea1 edema. There were no signs of intraocular inflammation. Histologic examination showed, as in previous studies (Ramirez et al., 1986), that the continuous elevation of IOP produced cupping of the optic nerve head (Figs 1 and 2). Normal rabbit eyes very often have cupping of the optic nerve head in view of the absence of lamina cribosa. Demarcated by arrows, the cupping we observed in glaucomatous eyes was not a normal variation because of the following characteristics: (1) it extended through the entire optic nerve head; and (2) excavation began at the margin of the retina. Another important finding that supported the diagnosis of the optic nerve cupping was the presence of hypertrophied astrocytes in the optic nerve of glaucomatous eyes (Fig. 3). These enlarged astrocytes, called genistiocytic astrocytes, usually occur as a glial reaction at sites of injury. The intraocular volume of the AH from glaucomatous eyes was significantly higher than in normal eyes (0.9 * 0.1, n = 12 vs 0.3 f 0.1. n = 12; P < 0.001). Immunoreactive ANF was demonstrated by direct RIA before and after extraction by Sep-Pak cartridges. In both cases, a closed parallelism was observed between various dilutions of AH or its extracts and the standard curve, indicating that ANF present in AH is indistinguishable from the peptide used for preparation of standard curve. The ANF concentration in AH from normal eyes was within the detection limit of the RIA whereas ANF levels in AH from glaucomatous eyes were significantly higher (Table 1). In 4 of the 16 rabbits injected with a-chymotrypsin, IOP and intraocular volume were similar to eyes treated with saline alone (12.0 f 1.6 mmHg and 0.3 * 0.1 ml, respectively). Histologically, the optic nerve head was no different from that of control eyes. ANF concentrations in AH from glau~omatous eyes did not differ from the values obtained for control eyes.
Fig. I, Light photomicrograph
of physiologic cup of the normal optic disc (arrows) (hematoxylin-eosin x IO) (R: retina; S: sclera).
Fig. 2. Light photomicrograph
of glaucomatous cupping of optic disc (arrows) (hematoxylin-eosin, (R: retina; S: sclera).
1307
x 10)
Fig.
3. Hypertrofied
astrocytes
(arrows)
in the optic nerve of giaucomatous x 120).
1308
eyes (hematoxylin-e
:osin.
ANF increases in aqueous humor of experimental glaucoma DISCUSStON
of our knowledge, this is the first of immunoreactive ANF in AH. Its origin in AH is not yet known. Stone and Glembotski (1986) identified immunoreactive ANF in the rat anterior uvea (iris and ciliary body) and retina. They demonstrated, by gel filtration chromatography and reverse phase high performance liquid chromatography that the major forms of the ANF-related material in the uvea and retina have molecular weights similar to the hypothalamic and plasma forms (ANF-28), clearly distinguishable from the larger cardiac form. We have detected immunoreactive ANF in the rabbit ciliary body, retina and choroid (unpublished results). However, because of the paucity of material, we were not able to analyze their molecular forms. In AH from glaucomatous eyes with moderately high IOP, we noted significantly increased ANF concentrations, a finding that suggests an important physiological role of ANF in the maintenance of IOP. This hypothesis is reinforced by data on the effects of ANF on IOP. Sugrue and Viader (1986) have demonstrated that topical, intracameral and intravitreal application of ANF lowers IOP by l&25% in ANF application, rabbits. By intravitreal Nathanson et al. (1987) induced a marked decrease of IOP which lasted for more than 48 hr. However, Mittag et al. (1987) found no measurable effects on IOP during 16-72 hr post-intravitreal injection. Whether these differences are attributable to the methods employed needs to be established. Further support for our hypothesis is found in the presence of ANF receptors in ciliary processes (Quirion, et al., 1984; Bianchi et al., 1986). Furthermore, ANF stimulated guanylate cyclase in membranes derived from ciliary processes (Mittag et al., 1987). Injection of a-chymotrypsin into the posterior chamber of rabbit eyes produced a significant elevation of IOP. The abnormalities we observed in the optic nerve and retina were similar to those reported by other authors (Vareilles et al., 1979; Hayreh, 1978). The cupping of the optic disc that we detected in glaucomatous eyes (Fig. 2) had the principal characteristics described by Vareilles et al. (1979): (1) total disappearance of the optic nerve head fibres; and (2) excavation beginning at the margin of the retina. These characteristics permit differentiation of glaucomatous cupping To the best
demonstration
1309
from the normal cupping that some rabbit eyes present in view of the absence of lamina cribosa. The genistiocytic astrocytes in the injured areas of the optic nerve (Fig. 3) could be considered a glial reaction to ocular hypertension. Of the rabbit eyes treated with a-chymotrypsin, 25% did not show any increase of IOP. This has also been observed by other authors (Vareilles et al., 1979; Best, Rabinovitz & Masket, 1975) and can be interpreted in two ways: (1) an error was made when injecting the drug; and (2) the rabbits did not respond to a-chymotrypsin. However, since ANF concentration in AH in these eyes was similar to eyes injected with saline, a-chymotrypsin per se may not be responsible for the increase of ANF in AH. The volume distension which occurs in the rabbit eyes with high IOP and intraocular volume could be a stimulus of ocular ANF secretion. It is well known that distension of the atria is a major stimulus of ANF release (Lang, Thoelken, Ganten, Luft, Rushkoaho & Unger, 1985). The increased or compensatory ANF concentration in eyes with high IOP could also be interpreted as a physiological response causing lower IOP as several authors have demonstrated that ANF application decreases IOP (Sugrue & Viader, 1986; Nathanson et al., 1987). It appears reasonable to believe that, because of the ANF’s hemodynamic actions, its increased concentration in AH from glaucomatous eyes could be a regulatory mechanism to prevent further deleterious effects of high IOP. In conclusion, this study demonstrates the presence of ANF in AH from rabbit eyes. Higher levels of ANF are evident in AH from glaucomatous eyes coinciding with high IOP. Acknowledgemenrs-This work was supported in part by FISS (No. 86/650), Foundation Fernandez-Cruz and the Medical Research Council of Canada (by a grant to Jolanta Gutkowska) (MA 10337) grants.
REFERENCES Best, M., Rabinovitz, A. Z. & Masket, S. (1975). Experimental achymotrypsin glaucoma. Anna/s of Ophrhalmology, 7, 803-8 10. Bianchi, C., Anand-Srivastava, M. B., de Lean, A., Gutkowska, J., Forthomme, D., Genest, J. & Cantin, M. (1986). Localization and characterization of specific receptors for atria1 natriuretic factor in the ciliary processes of the eye. Current Eye Research, 5, 283-293. de Bold, A. J. & Flynn, T. (1983). Cardionatrin I. A novel heart peptide with potent diuretic and natriuretic properties. Life Science, 33, 297-303.
RAQUELFERNANDEZ-DURAN(;O er al
1310
Gutkowska. J.. Bonan, R., Roy, D., Bourassa, M.. Garcia, R., Thibault, G., Genest, J. & Cantin. M. (1986). Atrial natriuretic factor in human plasma. Eiochemrcal and Biophysical
Research Communications,
139, 287-295.
Gutkowska, J. (1987). Radioimmunoassay for atrial natriuretic factor. Nuclear Medicine and Biology. 14, 323.-33 I. Haynh, S. (1978). Pathogenesis of optic nerve damage and visual field defects. In Glaucoma, conceprions q/a disease. Stuttgart, Thieme. Lang, R. E., Thoelken, H., Ganten, D.. Luft. F C.. Rushoaho, H. & Unger, T. H. (1985). Atrial natriuretic factor-a circulating hormone stimulated by volume loading. Nature, London, 314, 264266. Mittag, T. W., Tormay, A., Ortega, M. & Severin, C. (1987). Atrial natriuretic peptide (ANP), guanylate cyclase, and intraocular pressure in the rabbit eye. Current E_ve Research, 6, I 189-l 196. Nathanson, J. A., Hunnicutt. E. & Owen, C. (1987). Atriopeptins in the eye: Receptors, sound messengers and effects on intraocular pressure. lnresrigarive Ophthalmology and V&al
Science, 28(3),
273.
Quirion, R., Dalpe, M.. DeLean, A. Gutkowska. J., Canttn. M. & Genest. J. (1984). Atrial natriuretic factor (ANibinding sites in brain and related structures. Pep/ides. 5. 1167-1172 Ramirez, J. M.. Trtvitio, A. & Garcia, J, ( 1986). Anahsts de las alteraciones producidas en el glaucoma experimental a nivel de la cabeza del nervio optico: Estudio histologico. Archives
de la Sociedad
Espaiiola
de Ofialmologia
.(I.
291 296.
Stone. R. A. & Glembotskt, C. c’ (1986). Immunoreactive atrial natriuretic peptide in the rat eye: Molecular forms in anterior uvea and retina. Biochemical and Biophysical
Research
Communications.
134,
1022
1028.
Sugrue, M. F. & Viader, M. P. (1986). Synthetic atrial natriuretic factor lowers rabbit intraocular pressure. European Journal
of Pharmacology,
130. 349-350.
Vareilles, P., Durand, G., Siou, G. & Le Douarec. 3. C. (I 979). Le modele de glaucome experimental I I’a-chymotrypsin chez Ie lapin: Etude histologique. JOWMI Francalc d’Ophtalmologie.
2. 561-568.
Copyright
~2-69~9/90 $3.00 + 0.00 Q 1990 Pergamon Press pit
IMMUNOREACTIVE ATRIAL NATRIURETIC FACTOR IN AQUEOUS HUMOR: ITS CONCENTRATION IS INCREASED WITH HIGH INTRAOCULAR PRESSURE IN RABBIT EYES RAQUEL FER~A~DEZ-DURAN~,’ ALBERTOTRIVIIQO,’JOSEM. RAMIREZ,* MARIO GARCIA DE LACOBA,’ ANA RAMIRE&* JUAN J. SALAZAR,’ ARTURO FBBNANDEZ-CRUZ’ and JOLANTAGUTKOWSKA’ ‘Diabetic, Hypertension and Obesity Unit and 2Department of Ophthalmology, Laboratory of Ocular Morphology, Hospital Universitario San Carlos, Ciudad Universitaria, 28040-Madrid, Spain and 31nstitut de Recherches Cliniques de Montreal, I10 Avenue des Pins Ouest, Montreal, Canada H2W 1R7 (Received 9 February 1989; in revisedform
25 October 1989)
Abstract-Atria1 natriuretic factor (ANF) concentration in the aqueous humor (AH) was studied in rabbits with experimental glaucoma induced by injecting a-chymotrypsin into the posterior chamber. In normal rabbit eyes, the ANF concentration in AH was 3.1 f 1.2 pg/ml (% & SEM; n = t2), ranging from 0 to 5.8 pg/ml, whereas it was si~i~cantly higher in AH from glaucomatous rabbit eyes, being 8 1.O+ 9.8 pg/ml (n = 12). These findings were correlated with intraocular pressure (IOP), which was 13.0 + 2.4 mmHg (n = 12) in normal rabbit eyes and significantly greater in glaucomatous eyes: 24.4 f 3.0 mmHg (n = 12). Our data indicate that enhanced ANF release in AH during experimental glaucoma may play an important physiological role in modulating IOP. Aqueous humor
Atria1 natriuretic factor
Experimental glaucoma
INTRODUCTION
A great deal of evidence suggests that atria1 natriuretic factor (ANF) modulates body volume-salt homeostasis (de Bold & Flynn, 1983). Originally found in mammalian atria, ANF is not exclusively an atria1 hormone. Its presence has been demonstrated in many other tissues, such as heart ventricles, lung, various parts of the brain, anterior uvea and retina as well as blood and ascitic fluid. Immunoreactive ANF concentrations in the anterior uvea and retina of the rat are 31 and 8 rig/g tissue, respectively (Stone & Glembotski, 1986). A high density of ANF receptors has been observed on the epithelial side of the ciliary body (Quirion, Dalpe, DeLean, Gutkowska, Cantin & Genest, 1984; Bianchi, Anand-Srivastava, de Lean, Gutkowska, Forthomme, Genes1 & Cantin, 1986). Guanylate cyclase, the proposed transducer of the ANF signal to the intracellular compartment, is stimulated by ANF in membranes derived from ciliary processes (Mittag, Tot-may, Ortega & Severin, 1987). Furthermore, ANF has been shown to decrease intraocular pressure (IOP) in numerous species (Sugrue & Viader, 1986; Nathanson, Hunnicutt
Intraocular pressure
& Owen, 1987). Since IOP is increased in glaucoma, we decided to study the probable presence of ANF in ~aucomatous eyes to evaiuate the hypothesis that it may modulate IOP. MATERlALS
Experimental
AND METHODS
glaucoma
Adult, male, New Zealand albino rabbits (3.0-3.5 kg) were maintained under alternating 12-hr periods of darkness and light on laboratory chow and water ad libitum. Experimental glaucoma was produced in the right eye by injecting 0.86mg of ~-chymotrypsin diluted in 0.13 ml of saline into the posterior chamber according to a modification (Ramirez, Triviiio & Garcia, 1986) of the method of Vareilles, Durand, Siou and Le Douarec (1987). The left eye was injected with saline. IOP was measured bilaterally by aplanation tonometry. A local anesthetic (0.2% fluorescein-0.4% benoxinate) was given topically, and the tonometer (Draeger, Moller Wedel, Germany) was applied tangentially to the cornea for 5 sec. 40 days after the a-chymotrypsin injection, the animals were anesthetized by intravascular administration of
1305
RAQLIELFERNANDEZ-DWWW et ai
1306
sodium pentobarbital (30 mg/kg) and AH was drawn with a 1.0 ml-syringe. The needle (dia. 0.3 mm) was forced through the cornea1 scleral junction into the anterior chamber. The rabbits were then killed by anesthetic overdose and their eyes were immediately enucleated for dissection, which was followed by embedding in paraplast (Sherwood Medical, Ireland) and staining with hematoxylin-eosin. Measuremenr
of ANF
The aqueous humor (AH) samples were collected in chilled tubes containing protease inhibitors at the following final concentrations: 1 mg~m1 EDTA, 10e3 M phenylmethylsulfonyl fluoride (PMSF) (Sigma No. P-7626) and 5 x low6 M pepstatin A (Sigma No. P-4265). ANF extraction was performed according to the method used for plasma ANF (Gutkowska, Bonan, Roy, Bourassa, Garcia, Thibault, Genest & Cantin, 1986). Briefly, the Sep-Pak C-l 8 cartridges (Waters Associates, Milford, MA) were activated by washing with 8-10 ml acetonitrile then washed with 8-10ml ammonium acetate (0.2% at pH 4.0). The AH samples were then applied on the cartridges, washed with 5 ml ammonium acetate (0.2%, pH 4.0) and the absorbed ANF was eluted with 3 ml acetonitrile (60%) in ammonium acetate (0.2%, pH 4.0). The organic solvent was evaporated under nitrogen stream followed by lyophiliration. The residue was taken up in RIA buffer (0.1 M phosphate, pH 7.4) containing 0.3% NaCl, 0.1% BSA and 0.1% Triton X- 100. Immunoreactive ANF was measured by a specific and sensitive radioimmunoassay (RIA), as previously described (Gutkowska, 1987). The detection limit was 0.75 pg with a within and between assay variability of 10.2 and 18.7%, respectively. The recovery of standard ANF (34.5 and 69pg/ml) added to pooled AH was 79.0 + 3.2% (n = 10). Data analysis
The statistical significance of the differences between groups was evaluated by Student’s f-test. RESULTS
IOP was increased in the eyes of 12 out of 16 rabbits as soon as 72 hr after the cc-chymotrypsin administration. After 40 days, it was significantly higher than in rabbit eyes injected with saline alone (Table I).
Table
1, lmmunoreactive ANF in AH and IOP d norma: and glaucomatous rabbit eyes ANF
Normal eyes (n = 12) Glaucomatous eyes (n =
@gimf)
3.1 f I.2
12) 8I .? + 9.8’
10P (mmHg)
13.0& 2.4 24.4f 3.0,
Values are given as means ,+ SEM statistically different from normal values, *P c. 0.05.
Mo~hologi~ally, eyes treated with the enzyme had a buphthalmic appearance with tears in the Descemet’s membrane and a discrete cornea1 edema. There were no signs of intraocular inflammation. Histologic examination showed, as in previous studies (Ramirez et al., 1986), that the continuous elevation of IOP produced cupping of the optic nerve head (Figs 1 and 2). Normal rabbit eyes very often have cupping of the optic nerve head in view of the absence of lamina cribosa. Demarcated by arrows, the cupping we observed in glaucomatous eyes was not a normal variation because of the following characteristics: (1) it extended through the entire optic nerve head; and (2) excavation began at the margin of the retina. Another important finding that supported the diagnosis of the optic nerve cupping was the presence of hypertrophied astrocytes in the optic nerve of glaucomatous eyes (Fig. 3). These enlarged astrocytes, called genistiocytic astrocytes, usually occur as a glial reaction at sites of injury. The intraocular volume of the AH from glaucomatous eyes was significantly higher than in normal eyes (0.9 * 0.1, n = 12 vs 0.3 f 0.1. n = 12; P < 0.001). Immunoreactive ANF was demonstrated by direct RIA before and after extraction by Sep-Pak cartridges. In both cases, a closed parallelism was observed between various dilutions of AH or its extracts and the standard curve, indicating that ANF present in AH is indistinguishable from the peptide used for preparation of standard curve. The ANF concentration in AH from normal eyes was within the detection limit of the RIA whereas ANF levels in AH from glaucomatous eyes were significantly higher (Table 1). In 4 of the 16 rabbits injected with a-chymotrypsin, IOP and intraocular volume were similar to eyes treated with saline alone (12.0 f 1.6 mmHg and 0.3 * 0.1 ml, respectively). Histologically, the optic nerve head was no different from that of control eyes. ANF concentrations in AH from glau~omatous eyes did not differ from the values obtained for control eyes.
Fig. I, Light photomicrograph
of physiologic cup of the normal optic disc (arrows) (hematoxylin-eosin x IO) (R: retina; S: sclera).
Fig. 2. Light photomicrograph
of glaucomatous cupping of optic disc (arrows) (hematoxylin-eosin, (R: retina; S: sclera).
1307
x 10)
Fig.
3. Hypertrofied
astrocytes
(arrows)
in the optic nerve of giaucomatous x 120).
1308
eyes (hematoxylin-e
:osin.
ANF increases in aqueous humor of experimental glaucoma DISCUSStON
of our knowledge, this is the first of immunoreactive ANF in AH. Its origin in AH is not yet known. Stone and Glembotski (1986) identified immunoreactive ANF in the rat anterior uvea (iris and ciliary body) and retina. They demonstrated, by gel filtration chromatography and reverse phase high performance liquid chromatography that the major forms of the ANF-related material in the uvea and retina have molecular weights similar to the hypothalamic and plasma forms (ANF-28), clearly distinguishable from the larger cardiac form. We have detected immunoreactive ANF in the rabbit ciliary body, retina and choroid (unpublished results). However, because of the paucity of material, we were not able to analyze their molecular forms. In AH from glaucomatous eyes with moderately high IOP, we noted significantly increased ANF concentrations, a finding that suggests an important physiological role of ANF in the maintenance of IOP. This hypothesis is reinforced by data on the effects of ANF on IOP. Sugrue and Viader (1986) have demonstrated that topical, intracameral and intravitreal application of ANF lowers IOP by l&25% in ANF application, rabbits. By intravitreal Nathanson et al. (1987) induced a marked decrease of IOP which lasted for more than 48 hr. However, Mittag et al. (1987) found no measurable effects on IOP during 16-72 hr post-intravitreal injection. Whether these differences are attributable to the methods employed needs to be established. Further support for our hypothesis is found in the presence of ANF receptors in ciliary processes (Quirion, et al., 1984; Bianchi et al., 1986). Furthermore, ANF stimulated guanylate cyclase in membranes derived from ciliary processes (Mittag et al., 1987). Injection of a-chymotrypsin into the posterior chamber of rabbit eyes produced a significant elevation of IOP. The abnormalities we observed in the optic nerve and retina were similar to those reported by other authors (Vareilles et al., 1979; Hayreh, 1978). The cupping of the optic disc that we detected in glaucomatous eyes (Fig. 2) had the principal characteristics described by Vareilles et al. (1979): (1) total disappearance of the optic nerve head fibres; and (2) excavation beginning at the margin of the retina. These characteristics permit differentiation of glaucomatous cupping To the best
demonstration
1309
from the normal cupping that some rabbit eyes present in view of the absence of lamina cribosa. The genistiocytic astrocytes in the injured areas of the optic nerve (Fig. 3) could be considered a glial reaction to ocular hypertension. Of the rabbit eyes treated with a-chymotrypsin, 25% did not show any increase of IOP. This has also been observed by other authors (Vareilles et al., 1979; Best, Rabinovitz & Masket, 1975) and can be interpreted in two ways: (1) an error was made when injecting the drug; and (2) the rabbits did not respond to a-chymotrypsin. However, since ANF concentration in AH in these eyes was similar to eyes injected with saline, a-chymotrypsin per se may not be responsible for the increase of ANF in AH. The volume distension which occurs in the rabbit eyes with high IOP and intraocular volume could be a stimulus of ocular ANF secretion. It is well known that distension of the atria is a major stimulus of ANF release (Lang, Thoelken, Ganten, Luft, Rushkoaho & Unger, 1985). The increased or compensatory ANF concentration in eyes with high IOP could also be interpreted as a physiological response causing lower IOP as several authors have demonstrated that ANF application decreases IOP (Sugrue & Viader, 1986; Nathanson et al., 1987). It appears reasonable to believe that, because of the ANF’s hemodynamic actions, its increased concentration in AH from glaucomatous eyes could be a regulatory mechanism to prevent further deleterious effects of high IOP. In conclusion, this study demonstrates the presence of ANF in AH from rabbit eyes. Higher levels of ANF are evident in AH from glaucomatous eyes coinciding with high IOP. Acknowledgemenrs-This work was supported in part by FISS (No. 86/650), Foundation Fernandez-Cruz and the Medical Research Council of Canada (by a grant to Jolanta Gutkowska) (MA 10337) grants.
REFERENCES Best, M., Rabinovitz, A. Z. & Masket, S. (1975). Experimental achymotrypsin glaucoma. Anna/s of Ophrhalmology, 7, 803-8 10. Bianchi, C., Anand-Srivastava, M. B., de Lean, A., Gutkowska, J., Forthomme, D., Genest, J. & Cantin, M. (1986). Localization and characterization of specific receptors for atria1 natriuretic factor in the ciliary processes of the eye. Current Eye Research, 5, 283-293. de Bold, A. J. & Flynn, T. (1983). Cardionatrin I. A novel heart peptide with potent diuretic and natriuretic properties. Life Science, 33, 297-303.
RAQUELFERNANDEZ-DURAN(;O er al
1310
Gutkowska. J.. Bonan, R., Roy, D., Bourassa, M.. Garcia, R., Thibault, G., Genest, J. & Cantin. M. (1986). Atrial natriuretic factor in human plasma. Eiochemrcal and Biophysical
Research Communications,
139, 287-295.
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