- Email: [email protected]
Combined effect of forskolin and acetazolamide on intraocular pressure and aqueous flow in rabbit eyes
Exp. Eye Res. (1984) 39, 47-50
Combined Intraocular
Effect of Forskolin and Acetazolamide Pressure and Aqueous Flow in Rabbit JOSEPH
Department
CAPRIOLI
AND
MARVIN
on Eyes
SEARS
of Ophthalmology and Visual Science, Yale University Medicine, New Haven, CT 06510, U.S.A.
School of
(Received 12 September 1983 and accepted 27 September 1983, New York) Forskolin, a diterpene, a potent and direct stimulator of the catalytic unit of adenylatc cyclase, can reduce intraocular pressure when administered topically to rabbit eyes in a 1 o/0 suspension. Using an intravitreal plant of fluorescein-dextran, aqueous flow was measured by a modification of the method of Maurice. Forskolin reduces net aqueous flow by 61 y0 compared to the controls. Acetazolsmide (25 mg kg-i) administered intravenously reduces net aqueous flow by 45 %. Forskolin and acetazolamide together reduce net aqueous flow by 72 %. an important additive effect undoubtedly produced by different pathways. Key words : fomkolin ; rabbit eyes ; aqueous humor ; acetazolamide ; glaucoma ; eye pressure.
1. Introduction Forskolin reduces the intraocular pressure (IOP) of rabbits, monkeys and humans when applied as a topical suspension (Caprioli and Sears, 1983; Caprioli, Sears, of aqueous flow Bausher, Gregory and Mead, 1983). In those studies measurements in rabbits demonstrated a net decrease of 40-50 y. after a single topical drop of a 1.0 y. forskolin suspension. The present study utilizes a modification of a recently reported technique for the determination of aqueous flow (Maurice, 1983) to investigate the combined effects of forskolin and acetazolamide on net aqueous flow in rabbit eyes.
2. Materials
and Methods
Male New Zealand White rabbits weighing approximately 25 kg were used. Pure forskolin was obtained from HoechstiRoussel Pharmaceuticals, Inc. and from Calbiochem Behring Corporation (La Jolla, CA), fluorescein-dextran (MW 70600) from Sigma Chemical Corporation (St Louis, MO), and parenteral acetazolamide from Lederle Laboratories (Pearl River, NY). A 1.0% forskolin suspension was prepared in a balanced salt solution containing 65 y0 hydroxypropyl methylcellulose. Acetazolamide was prepared in distilled water for intravenous use. Two weeks prior to drug treatment 15 rabbits received bilateral intra-vitreal injections of fluoreaccin-dextran. Ten microliters of a 10% solution was injected into the midvitreous through the pars plana using a 30 gauge needle. There was no visible reflux of material from the injection site. One among 30 eyes became inflamed several days after the injection, and this animal was not tested. All other eyes remained white without irritation. Groups treated with acctazolamide only, forskolin only, a combination of acetazolamide and forskolin, and control talc were studied. IOP was measured at 0, 1, 2, 3, and 4 hr in awake animals after topical proparacaine using an applanation pneumotonometcr calibrated for rabbit eyes. Immediately after zero time IOP measurement, eight animals received intravenous acetazolamide, slowly (25 mg kg-‘). Six of these animals were treated at 1 hr after acetazolamide with 56 gl of a 1.0% topical forskolin suspension (on to the right Please address reprints and correspondenceto Dr Joseph Caprioli, Department of Ophthalmology and Visual Science, Yale University School of Medicine, 310 Cedar Street, P.O. Box 3333, New Haven, CT 06510, U.S.A. 001~35/84/019047+04
$03.00/0
@1984 Academic Press Inc. (London) Limited
48
J. CAPRIOLI
AIiD
MARVIS
SEARS
eye). An additional six rabbits were treated with 1 o/0 forskolin in the right eye. All left eyes of this last group received a drop of 1.0 o/0 talc suspension in the same vehicle as a control. Paracenteses were performed on each eye at 4 hr after administration of acetazolamide and/or 3 hr after topical forskolin: 100,~l of anterior chamber aqueous was removed. Fluorescein concentrations were determined spectrophotofluorometrically using a standard curve. In this way changes in aqueous flow relative to control eyes were calculated. It was assumed in these calculations that (1) the only exit route for fluorescein dextran from the eye is via normal aqueous outflow pathways. and that (2) the anterior chamber fluorescein concentration is linearly related to the inverse of aqueous flow during the interval of the
experiment (Maurice, 1983). All data are reported as rnean+~.~.~. Student’s t test.
(n). Statistical
analyses
were
conducted
using
3. Results Mean IOP measurements in all groups are displayed against time in Fig. 1. The IOP remained constant in the control group throughout the experiment. IOP was reduced significantly compared to controls 2 and 3 hr after topical forskolin alone (P< @Ol ).
I
2 Time
3
4
(hr)
FIG. 1. Intraocular pressure (mean+s.z.u.) versus time (hr) for control (g), acetazolamide-treated (@), forskolin-treated (El), and combined acetazolamide- and forskolin-treated groups (0). See text for description of significant differences.
Acetazolamide and combined acetazolamideand forekolin-treated groups showed significant reductions compared to control eyes at 1,2,3 and 4 hr after acetazolamide administration (P < @02). Significant differences were also found between acetazolamide versus combined acetazolamideand forskolin-treated groups; the eyes receiving combined treatment had lower IOPs at 2, 3 and 4 hr (P = @Ol, O-04 and @03, respectively). Mean anterior chamber fluorescein concentrations for each group, magnitudes of aqueous flow reductions and statistical results relative to the control group are given in Table I. Similar reductions in aqueous flow were found for the separately treated acetazolamide (45 %) and forskolin (51%) groups. The combination of the two drugs
AQUEOUS
FLOW
REDUCTION
BY FORSKOLIN
AND
ACETAZOLAMIDE
49
TABLET
Eyes @) Control Acetazolamide Forskolin Acetazolamide forskolin
6 10 6 and 6
Aqueous fluorescein concentration hi3 PI-‘) 3.05 ko.41 554+656 627 *w33 11.07* 1.61
Significance (P)* vs. acetazolamide
vs. control 0603 om6 < 0661
648 0009
V8. forskolin
Relative decrease in aqueous flow (%)
0025
45 51 72
Relative decreases in aqueous flow as determined by aqueous fluorescein concentration. Aqueous flow is inversely proportioned to aqueous fluorescein concentration (see test). * Results oft tests comparing aqueous fluorescein concentration in the different treatment groups,
produced
a significantly
greater
reduction
in aqueous flow than either
agent alone
(72%). 4. Discussion Stimulation of ciliary adenylate cyclase by cholera toxin (Gregory, Sears, Bausher, Mishima and Mead, 1981), by certain organic fluorides and by commercial preparations of gonadotropins (Sears and Mead, 1983) lowers IOP by reducing net aqueous inflow. Forskolin is a diterpene derivative of the plant Coleus forskohlii which stimulates adenylate cyclase without interacting with cell surface receptors, and lowers IOP in rabbits, monkeys and man (Caprioli and Sears, 1983; Caprioli et al., 1983; Sears, Caprioli, Kondo and Bausher, 1983). This report demonstrates the additive effects of acetazolamide and forskolin on IOP and aqueous flow in rabbits. IOP is reduced more by a combination of these drugs than by either drug alone. Modifying Maurice’s potentially exciting technique (1983), we have found that acetazolemide or forskolin independently reduces net aqueous inflow by approximately 59% and that a combination of these agents reduces flow by approximately 75 %. Since this work was done, using topically applied fluorescein and the ‘fluorotron’ scan, Bartels and Neufeld [in rabbits, Vis. Sci. ARVO SuppZ. (1984)], P. Lee et al. [in monkeys, Invest. Ophthulmol. (1984) in press] and Burstein, Sears and Mead [in humans, Exp. Eye Res. (1984) in press] have all found reductions in aqueous flow of 25, 34, and 35 y. respectively after topical forskolin. This additive effect on net aqueous flow by acetazolamide and forskolin is not surprising in view of the quite different pharmacologic action of each agent. How the tissue mechanism of each works to reduce flow is speculative. Acetazolamide may decrease aqueous inflow by limiting the accession rate of HCO, into the nascent posterior aqueous (Zimmerman, Garg, Vogh and Maren, 1976). Perhaps sodium is coupled to this process. Evidence pro and con has been well summarized elsewhere (Cole, 1966). Forskolin increases intracellular ciliary cyclic AMP by directly stimulating adenylate cyclase (Caprioli and Sears, 1983; Caprioli et al., 1983; Bausher, Gregory and Sears, 1983). The stimulation of adenylate cyclase by cholera toxin in diverse cellular systems - choroid plexus, cochlear epithelium, intestinal epithelium promotes fluid and electrolyte movement from the basal (serosal) side of the cell out through the cellular apex (mucosa) (Field, 1971: Feldman and Brusilow, 1976:
50
.J.(‘APKIOI,l
AND
Jl~iRVIN
SEARS
Epstein, Feldman and Brusilow, 1977). 1n wrtain cases an increase in the apical permeability to chloride ion may o(lcur (Frizzell. Field and Schultz. 1979) as in the instance of the cornea (Klyce and Wong, 1977). Aw a result of the invagination of thp optic vesicle, the basal side of the ciliary non-pigment,ed epithelium borders the posterior chamber. Thus, activation of adenylate cyrlase in this tissue by forskolin may promote reabsorption of fluid from the posterior chamber and secretion int)o the ciliary stroma, thereby reducing ‘net’ aqueous inflow (Fujita, Kondo and Sears, 1984, unpubl. obs. ; Sears et, al., 198313). Vascular effects, secondary to increased ciliary body blood flow (Caprioli et al.. 1983a; Sears et al.. 1983b) cannot yet he excluded. Forskolin represents a potential new drug for the treatment of glaucoma. Forskolin may be especially useful in combination with other currently used drugs because of its unique mode of action. ACKNOWLEDGMENTS This work was supported by Research to Prevent Blindness, Inc., The New Haven Foundation, Foresight, Inc., and CT.S. Public Health Service grants EY 00785 and EY 00237. We thank Alden Mead for his expert technical assistance. REFERENCES Bausher, L., Gregory, D. and Sears, M. (1983). Forskolin activates adenylate cyclase in ciliary processes. Invest. Ophthal. I/is. Sci. (Suppl.) 24, 4. Caprioli, J. and Sears, M. (1983). Forskolin lowers intraocular pressure in rabbits, monkeys, and man. The Lancet i (8331), 958-9. Caprioli, J., Sears, M., Bausher, L., Gregory, D. and Mead, A. (1984). Stimulation of ciliary adenylate cyclase by forskolin lowers intraocular pressure by reducing net aqueous humor inflow. Invest. Ophth.aEmol. 1’i.s. Sci. 25. 267-77. Cole, D. F. (1966). Aqueous humour formation. Dot. Ophthulmol. 21, 1 l&238. Epstein, M. D., Feldman, A. M. and Brusilow, S. W. (1977). Cerebrospinal fluid production: stimulation by cholera toxin. Science 196, 1012-13. Feldman, A. M. and Brusilow, S. W. (1976). Effects of cholera toxin in cochlear endolymph production: model for endolymphatic hydrops. Pot. Nat1 Acad. Sri. U.S.A. 73, 1761-4. Field, M. (1971). Ion transport in the rabbit ileal mucosa. II. Effects of cyclic AMP. Am. .I. Physiol. 221, 992-7. Frizzell, R. A., Field, M. and Schultz, S. G. (1979). Sodium-coupled chloride transport by epithelial tissues. Am. J. Physiol. 236, Fl-F8. Gregory, D., Sears, M., Bausher, L., Mishima, H. and Mead, A. (1981). Intraocular pressure and aqueous flow are decreased by cholera toxin. Invest. Ophthalmol. Vis. Sci. 20, 311-81. Klyce, S. D. and Wong, R. K. S. (1977). Site and mode of adrenalin action on chloride transport across the rabbit cornea1 epithelium. J. Physiol. 266, 777-99. Maurice, D. (1983). A simple method for measuring aqueous flow in the rabbit. Invest. Ophthalmol. Vis. Sci. (Suppl.) 24, 5. Sears, M. D. and Mead, A. (1983). A major pathway for the regulation of intraocular pressure. Znternatl. Ophthalmol. I, 201-12. Sears, M., Caprioli, J., Kondo, K. and Bausher, L. (1983). A mechanism for the control of aqueous humor formation. In Applied Pharmacology in the Medical Treatment of the Glaucomas (Eds Drance, S. and Neufeld, A.).New York, Grune and Stratton. (In press.) Zimmerman, T. J., Garg, L. C., Vogh, B. P. and Maren, T. H. (1976). The effect of acetazolamide on the movements of anions into the posterior chamber of the dog eye. J. Pharmawl. Exp. Ther. 196, 510-16.
Combined Intraocular
Effect of Forskolin and Acetazolamide Pressure and Aqueous Flow in Rabbit JOSEPH
Department
CAPRIOLI
AND
MARVIN
on Eyes
SEARS
of Ophthalmology and Visual Science, Yale University Medicine, New Haven, CT 06510, U.S.A.
School of
(Received 12 September 1983 and accepted 27 September 1983, New York) Forskolin, a diterpene, a potent and direct stimulator of the catalytic unit of adenylatc cyclase, can reduce intraocular pressure when administered topically to rabbit eyes in a 1 o/0 suspension. Using an intravitreal plant of fluorescein-dextran, aqueous flow was measured by a modification of the method of Maurice. Forskolin reduces net aqueous flow by 61 y0 compared to the controls. Acetazolsmide (25 mg kg-i) administered intravenously reduces net aqueous flow by 45 %. Forskolin and acetazolamide together reduce net aqueous flow by 72 %. an important additive effect undoubtedly produced by different pathways. Key words : fomkolin ; rabbit eyes ; aqueous humor ; acetazolamide ; glaucoma ; eye pressure.
1. Introduction Forskolin reduces the intraocular pressure (IOP) of rabbits, monkeys and humans when applied as a topical suspension (Caprioli and Sears, 1983; Caprioli, Sears, of aqueous flow Bausher, Gregory and Mead, 1983). In those studies measurements in rabbits demonstrated a net decrease of 40-50 y. after a single topical drop of a 1.0 y. forskolin suspension. The present study utilizes a modification of a recently reported technique for the determination of aqueous flow (Maurice, 1983) to investigate the combined effects of forskolin and acetazolamide on net aqueous flow in rabbit eyes.
2. Materials
and Methods
Male New Zealand White rabbits weighing approximately 25 kg were used. Pure forskolin was obtained from HoechstiRoussel Pharmaceuticals, Inc. and from Calbiochem Behring Corporation (La Jolla, CA), fluorescein-dextran (MW 70600) from Sigma Chemical Corporation (St Louis, MO), and parenteral acetazolamide from Lederle Laboratories (Pearl River, NY). A 1.0% forskolin suspension was prepared in a balanced salt solution containing 65 y0 hydroxypropyl methylcellulose. Acetazolamide was prepared in distilled water for intravenous use. Two weeks prior to drug treatment 15 rabbits received bilateral intra-vitreal injections of fluoreaccin-dextran. Ten microliters of a 10% solution was injected into the midvitreous through the pars plana using a 30 gauge needle. There was no visible reflux of material from the injection site. One among 30 eyes became inflamed several days after the injection, and this animal was not tested. All other eyes remained white without irritation. Groups treated with acctazolamide only, forskolin only, a combination of acetazolamide and forskolin, and control talc were studied. IOP was measured at 0, 1, 2, 3, and 4 hr in awake animals after topical proparacaine using an applanation pneumotonometcr calibrated for rabbit eyes. Immediately after zero time IOP measurement, eight animals received intravenous acetazolamide, slowly (25 mg kg-‘). Six of these animals were treated at 1 hr after acetazolamide with 56 gl of a 1.0% topical forskolin suspension (on to the right Please address reprints and correspondenceto Dr Joseph Caprioli, Department of Ophthalmology and Visual Science, Yale University School of Medicine, 310 Cedar Street, P.O. Box 3333, New Haven, CT 06510, U.S.A. 001~35/84/019047+04
$03.00/0
@1984 Academic Press Inc. (London) Limited
48
J. CAPRIOLI
AIiD
MARVIS
SEARS
eye). An additional six rabbits were treated with 1 o/0 forskolin in the right eye. All left eyes of this last group received a drop of 1.0 o/0 talc suspension in the same vehicle as a control. Paracenteses were performed on each eye at 4 hr after administration of acetazolamide and/or 3 hr after topical forskolin: 100,~l of anterior chamber aqueous was removed. Fluorescein concentrations were determined spectrophotofluorometrically using a standard curve. In this way changes in aqueous flow relative to control eyes were calculated. It was assumed in these calculations that (1) the only exit route for fluorescein dextran from the eye is via normal aqueous outflow pathways. and that (2) the anterior chamber fluorescein concentration is linearly related to the inverse of aqueous flow during the interval of the
experiment (Maurice, 1983). All data are reported as rnean+~.~.~. Student’s t test.
(n). Statistical
analyses
were
conducted
using
3. Results Mean IOP measurements in all groups are displayed against time in Fig. 1. The IOP remained constant in the control group throughout the experiment. IOP was reduced significantly compared to controls 2 and 3 hr after topical forskolin alone (P< @Ol ).
I
2 Time
3
4
(hr)
FIG. 1. Intraocular pressure (mean+s.z.u.) versus time (hr) for control (g), acetazolamide-treated (@), forskolin-treated (El), and combined acetazolamide- and forskolin-treated groups (0). See text for description of significant differences.
Acetazolamide and combined acetazolamideand forekolin-treated groups showed significant reductions compared to control eyes at 1,2,3 and 4 hr after acetazolamide administration (P < @02). Significant differences were also found between acetazolamide versus combined acetazolamideand forskolin-treated groups; the eyes receiving combined treatment had lower IOPs at 2, 3 and 4 hr (P = @Ol, O-04 and @03, respectively). Mean anterior chamber fluorescein concentrations for each group, magnitudes of aqueous flow reductions and statistical results relative to the control group are given in Table I. Similar reductions in aqueous flow were found for the separately treated acetazolamide (45 %) and forskolin (51%) groups. The combination of the two drugs
AQUEOUS
FLOW
REDUCTION
BY FORSKOLIN
AND
ACETAZOLAMIDE
49
TABLET
Eyes @) Control Acetazolamide Forskolin Acetazolamide forskolin
6 10 6 and 6
Aqueous fluorescein concentration hi3 PI-‘) 3.05 ko.41 554+656 627 *w33 11.07* 1.61
Significance (P)* vs. acetazolamide
vs. control 0603 om6 < 0661
648 0009
V8. forskolin
Relative decrease in aqueous flow (%)
0025
45 51 72
Relative decreases in aqueous flow as determined by aqueous fluorescein concentration. Aqueous flow is inversely proportioned to aqueous fluorescein concentration (see test). * Results oft tests comparing aqueous fluorescein concentration in the different treatment groups,
produced
a significantly
greater
reduction
in aqueous flow than either
agent alone
(72%). 4. Discussion Stimulation of ciliary adenylate cyclase by cholera toxin (Gregory, Sears, Bausher, Mishima and Mead, 1981), by certain organic fluorides and by commercial preparations of gonadotropins (Sears and Mead, 1983) lowers IOP by reducing net aqueous inflow. Forskolin is a diterpene derivative of the plant Coleus forskohlii which stimulates adenylate cyclase without interacting with cell surface receptors, and lowers IOP in rabbits, monkeys and man (Caprioli and Sears, 1983; Caprioli et al., 1983; Sears, Caprioli, Kondo and Bausher, 1983). This report demonstrates the additive effects of acetazolamide and forskolin on IOP and aqueous flow in rabbits. IOP is reduced more by a combination of these drugs than by either drug alone. Modifying Maurice’s potentially exciting technique (1983), we have found that acetazolemide or forskolin independently reduces net aqueous inflow by approximately 59% and that a combination of these agents reduces flow by approximately 75 %. Since this work was done, using topically applied fluorescein and the ‘fluorotron’ scan, Bartels and Neufeld [in rabbits, Vis. Sci. ARVO SuppZ. (1984)], P. Lee et al. [in monkeys, Invest. Ophthulmol. (1984) in press] and Burstein, Sears and Mead [in humans, Exp. Eye Res. (1984) in press] have all found reductions in aqueous flow of 25, 34, and 35 y. respectively after topical forskolin. This additive effect on net aqueous flow by acetazolamide and forskolin is not surprising in view of the quite different pharmacologic action of each agent. How the tissue mechanism of each works to reduce flow is speculative. Acetazolamide may decrease aqueous inflow by limiting the accession rate of HCO, into the nascent posterior aqueous (Zimmerman, Garg, Vogh and Maren, 1976). Perhaps sodium is coupled to this process. Evidence pro and con has been well summarized elsewhere (Cole, 1966). Forskolin increases intracellular ciliary cyclic AMP by directly stimulating adenylate cyclase (Caprioli and Sears, 1983; Caprioli et al., 1983; Bausher, Gregory and Sears, 1983). The stimulation of adenylate cyclase by cholera toxin in diverse cellular systems - choroid plexus, cochlear epithelium, intestinal epithelium promotes fluid and electrolyte movement from the basal (serosal) side of the cell out through the cellular apex (mucosa) (Field, 1971: Feldman and Brusilow, 1976:
50
.J.(‘APKIOI,l
AND
Jl~iRVIN
SEARS
Epstein, Feldman and Brusilow, 1977). 1n wrtain cases an increase in the apical permeability to chloride ion may o(lcur (Frizzell. Field and Schultz. 1979) as in the instance of the cornea (Klyce and Wong, 1977). Aw a result of the invagination of thp optic vesicle, the basal side of the ciliary non-pigment,ed epithelium borders the posterior chamber. Thus, activation of adenylate cyrlase in this tissue by forskolin may promote reabsorption of fluid from the posterior chamber and secretion int)o the ciliary stroma, thereby reducing ‘net’ aqueous inflow (Fujita, Kondo and Sears, 1984, unpubl. obs. ; Sears et, al., 198313). Vascular effects, secondary to increased ciliary body blood flow (Caprioli et al.. 1983a; Sears et al.. 1983b) cannot yet he excluded. Forskolin represents a potential new drug for the treatment of glaucoma. Forskolin may be especially useful in combination with other currently used drugs because of its unique mode of action. ACKNOWLEDGMENTS This work was supported by Research to Prevent Blindness, Inc., The New Haven Foundation, Foresight, Inc., and CT.S. Public Health Service grants EY 00785 and EY 00237. We thank Alden Mead for his expert technical assistance. REFERENCES Bausher, L., Gregory, D. and Sears, M. (1983). Forskolin activates adenylate cyclase in ciliary processes. Invest. Ophthal. I/is. Sci. (Suppl.) 24, 4. Caprioli, J. and Sears, M. (1983). Forskolin lowers intraocular pressure in rabbits, monkeys, and man. The Lancet i (8331), 958-9. Caprioli, J., Sears, M., Bausher, L., Gregory, D. and Mead, A. (1984). Stimulation of ciliary adenylate cyclase by forskolin lowers intraocular pressure by reducing net aqueous humor inflow. Invest. Ophth.aEmol. 1’i.s. Sci. 25. 267-77. Cole, D. F. (1966). Aqueous humour formation. Dot. Ophthulmol. 21, 1 l&238. Epstein, M. D., Feldman, A. M. and Brusilow, S. W. (1977). Cerebrospinal fluid production: stimulation by cholera toxin. Science 196, 1012-13. Feldman, A. M. and Brusilow, S. W. (1976). Effects of cholera toxin in cochlear endolymph production: model for endolymphatic hydrops. Pot. Nat1 Acad. Sri. U.S.A. 73, 1761-4. Field, M. (1971). Ion transport in the rabbit ileal mucosa. II. Effects of cyclic AMP. Am. .I. Physiol. 221, 992-7. Frizzell, R. A., Field, M. and Schultz, S. G. (1979). Sodium-coupled chloride transport by epithelial tissues. Am. J. Physiol. 236, Fl-F8. Gregory, D., Sears, M., Bausher, L., Mishima, H. and Mead, A. (1981). Intraocular pressure and aqueous flow are decreased by cholera toxin. Invest. Ophthalmol. Vis. Sci. 20, 311-81. Klyce, S. D. and Wong, R. K. S. (1977). Site and mode of adrenalin action on chloride transport across the rabbit cornea1 epithelium. J. Physiol. 266, 777-99. Maurice, D. (1983). A simple method for measuring aqueous flow in the rabbit. Invest. Ophthalmol. Vis. Sci. (Suppl.) 24, 5. Sears, M. D. and Mead, A. (1983). A major pathway for the regulation of intraocular pressure. Znternatl. Ophthalmol. I, 201-12. Sears, M., Caprioli, J., Kondo, K. and Bausher, L. (1983). A mechanism for the control of aqueous humor formation. In Applied Pharmacology in the Medical Treatment of the Glaucomas (Eds Drance, S. and Neufeld, A.).New York, Grune and Stratton. (In press.) Zimmerman, T. J., Garg, L. C., Vogh, B. P. and Maren, T. H. (1976). The effect of acetazolamide on the movements of anions into the posterior chamber of the dog eye. J. Pharmawl. Exp. Ther. 196, 510-16.