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Blockade of the ocular effects of acetazolamide by phencyclidine
Exp. Eye Res. (1977) 24, lS?-127
Blockade of the Ocular Effects of Acetazolamide by Phencyclidine
Phenryclidine. in low concentrations, has been found to block the vasoconstrictor action of acetazolamidc in t,he isolated, arterially perfused iris-ciliary body of the cat. Similar conwntrations of phenayclidine, in enwleated, arterially perfused whole cat eyes was found to prevent the action of acetazolamide to decrease the rate of aqueous humor tbrmat’ion. Esperiments in living rhesus monkeys also demonstrate that the inhibitory effects of acetnzolamide on aqueous humor formation cnn be effectively blocked by phencyclidiw. phrnryrlidine: vasoconstriction: t.at : Key words: aqueous humor: acetazolamide: monkey.
1. Introduction dcetazolamide has been demonstrated to produce vasoconstriction (Macri and Brown 1961; Macri and Cevario, 1975) and to decreasethe rate of aqueoushumor formation in the intact (Macri, Dixon and Rall, 1965; Oppelt, 1967) and cnucleated (Macri and (‘evario, 1975), arterially perfused cat eye. In the enucleated eye both of these actions are capable of being inhibited by the ganglionic blocking agent?hexamethonium (C-6) Macri and Ccvario, 1975). Blockage of acetazolamide action. hy hexamethonium has strengthened the concept that the action of acetazolamide is mediated by a local vascular constriction rather than by inhibition of carbonic anhydrase (Nacri acd Cevario: 19i5). It would be tlesiral)le to determine if a compound such as C-6, which has no known action on carbonic anhydrase, is also capable of blocking the action of acetazolamide on the eye of the anesthetized animal. Unfortunately, (~‘-6.is not amenable for this type stutly since its ganglionic blocking properties produce a marked decline of Moot I pressure which alters steady-state conditions of comparison. Fortuitously, another substance, phencpclidine, has been found to exhibit actions on the eye much like those of (‘-6. In the studies to be reported here. phencyclidine is shown to l)e a specific blocker of both the acetazolamide vascular and aqueous hunior activity in the cnucleated eye preparations. Phencyclidine is also demcnstrated to inhibit the actions of acetazolamide on aclueoushumor formation in the living rhesus monkey. 2. Methods Youug adult cats of either sex were utilized in this study. The armnalswere auesthetized with pentobarbital Na (30.0 mg/kg administered iutraperitoneallv). Ascorbic acid (‘LO.0 mg) was administeredin salinesolution, i.v. 5 min before the animal was killed and the eyes enucleated. The proceduresfor the preparation of the isolated, arterially perfusediris-ciliary body
and the whole cat eyes were identical to those previously reported (Macri and Brown, 1961; Macri and Cevario, 1973). Briefly, both preparations were perfusedintra-arterially with warmed (36°C) Eagles Basal Medium (EBM) at a pH of 7.4. The EBM solution was modified to contain 0.8% NaCl and 0.1 mg% ascorbic acid. No serum was used in the
F.
122
J.
MAC’XI
AA-I)
$.
.J. (‘I~V.-\IiIU
medium. Perfusate flow was measured utilizing a differential pressure-type tlowmeter (Macri and Brown, 1961). dqueous humor turnover in the enucleated, arterially perfusetl eyes \\eas determined by the method previously ut,ilized in the in viva cat (Jlacri il11cl O’Rourke, 1970). Male and female Mcccaca mzclattu (Rhesus) monkeys weighing between 2.0 and 3.5 kg were anesthetized with Sernylan (phencytlidine hydrochloride, 0.5 mg/kg). Small suhsequent doses of phencyclidine were given, when necessary, to maintain anesthesia. In a second series of experiments, Urethan (0.5 g/kg) was injected i.p. to maintain anesthesia, following the initial anesthetizing dose of phencyclidine. One femoral vein was cannulated in all monkeys for the administration of acetazolamide. The aqueous humor turnover in the living monkev was determined by the [14C]inulin dilution technique (Macri, Dixon and Rall, 1965)mo&fied asreported (Cevario and JIacri, 1974). Concentrations of drugs are expressed as sa1t.s. 3. Results Effects ia the iris-ciliury body preparation In seven experiments, phencyclidine (O-01 pg/ml) was perfused together with acetazolamide (20.0 pg/ml) through one long posterior ciliary artery of this preparation. Under this condition, vasoconstriction was not observed. Subsequent perfusion of the same preparations with acetazolamide alone, in all experiments, produced a decreasein arterial perfusion flow rate which averaged 3.12% (Table I).
Phencyclidine blockadeof acetazolamide vasoconstrictionin the isolated iris-cilia.ry body preparation
Control
Arterial perfusate Phenoyclidinef PO1 f&4
flow rate (~l/min) Acetazolamide (20.0 pg/ml)
*Meanfs.E.-seven preparations. tStatistically significant P < O-01 (paired
Acetazolamide WfJ I*@4
t-test).
Eflects on enucleated,arterially perfused cat eyes Six eyes were studied in this series(Table II). The rate of aqueoushumor formation was increased by the intra-arterial perfusion of Ach (O-01 rig/ml) +eserine (Es) (10.0 pg/ml). The formation rate of aqueous humor was found to increase from a basal level of 11.11. pl/min to 2’7.31 pl/ min. The addition of phencyclidine (0.01 pg/ml) to the perfusate media produced within 30 min a statistically significant, but small (3.7 &nin)
decrease in the aqueous
humor
inflow
rate. The addition
of acetazol-
amide (20.0 rug/ml) to the perfusion medium did not alter the rate of aqueous humor formation from those levels observed with phencyclidine alone. A previous report (Macri and Cevario, 1975) has demonstrated that the administration of acetazolamide to the Ach +Es stimulated eye, produced a 35% decreaseof the stimulated inflow rate. In a secondseriesof seven experiments (Table III), a different protocol was followed
INHIBITIOB
OF
ACETAZOLAMIDE
BY
123
PHEKC:TCLIDIXE
to verify the finding that phencyclidine could block the action of acetazolamide on aqueous humor production. After obtaining steady-state values of basal aqueous humor formation: the combination of aceta~olamide (20.0 mg/l) and phencyclidine
Block by phencyclidine of ncetazolamide inhibition of stinrulated aqueous humor form&ion in ellucleated arterially perfused cat eye (Series 1)
cbntro1
Il.11 (6) 2 141
Achl
l&wine
Inflow (rljmin) Ach +Eserine + Phencwlidinr
27.41 (s) *2.7&4
dch + Eserine 7 Phencyclidine + acetazolamide
23.63 (8) *7.2-l
-wean*S.E. Number of eyes shown in brackets. (R) Statistically significant P < 0.05 paired l-test. (n.s.) Not statistically significant. Ach 0.01 np/ml+eserine 10.0 pg/ml: phencyclidine
23.61 (n.8.) f%lX
O+l
&ml:
scetazolamide
20.0 pg/ml.
(0.01 pg/ml) were perfused first. No significant effect on basal levels of aqueous humor formation was noted. Subsequent arterial perfusion of the above drugs plus the stimulants Sch (0.01 rig/ml) and Es (10.0 pg/ml) produced a statistically significant, increase in the rate of aqueous humor formed (17.23 pl/min to 44.47 pl/min). TABLE
III
Block by phencyclidine of ncetazolnmide inhibition of stimulated rrqueow humorformation in enucleated arterially perfused cat eye (Series 2) Inflow
(‘ontrol 17,65(7) :t l%J
Acetazolamide +phencyclidine 17.23 (n.5.) j1.70
(pljmin) Acetazolamide +phencyclidine +Ach+eserine 44.47 +5~83
Mean&s.& Kumber of eyes shown in brackets. (8) Statistically significant P < 0.05, paired f-test. (n.s.) Not statistically significant. Ach 0.01 ng/ml+eserine 10.0 pz/mi: phencyclidine
(s)
0.01 &ml;
Acetazolamidcf Achfrswine 21.60
(s)
*“.jz
acctazolamide
204 pa/ml.
Alternating the arterial perfusate to another solution which did not contain the blocking agent phencyclidine, but had identical concentrations of Ach, Es and acetazolamide caused a 51% reduction in the rate of aqueous humor formation (from 44.47 @nin to 21.60 &‘min). E$ects on aqueoushumor production
i,n
anesthetizedrhesus,monkeys
A previous study of the rhesusmonkey (Cevario and Macri, 1974), hasdemonstrated that the rate of aqueous humor formation remains identical when anesthesia is induced by phencyclidine and maintained with either phencyclidine or urethan.
124
F. ,J. MAC’RI
ASI)
F. 5. (.‘EVAHIO
Ten experinwnts were pcrformetl on rhesus monkeys in which the ancst,hesial was maintained with w&an after incluction with phencyclidine (Table I\?). At the rw.l of 1 hr. at which time the eflects of l~henc~clidinr wrct presumably tlissipatetl ((‘hen, Xnsor, Ruswl ancl Rohner. 1959). tllp rate of i~ql~eoushw~lor fortl&on \vas clrtermined in one ttye for one how tinir lwriod. The average valw 0l)taitrctl for this il,
Actdo>lsof ncetctzolawide
on aqurous
hwror
fovwaatio~~ i,n the desus monkey
control interval was 3.09 pl/min. Acetazolamidc (50-100 mg/kg i-v.) was then administered to each monkey and following a 30 min equilibration period, aqueous humor production was determined for a 1 hr interval in the second eye. Acetazolamide was observed to decreaset)hc mtc of aqueoushumor formation by 1.64 pljniin to a value of 145 pl/min. In a second seriesof 10 rhesus monkeysi anesthesia was induced and maintained with phencyclidine alone. The sameprotocol was followed as above except that the dose of acctazolamide was increawtl to 200&300 nlg/kg. Control values of aqueous humor formation averaged 3.01 $jmin, which was not statistically different from control values of the urethane supplemented animals. Acetazolamide administration causeda 0.42 ~ljmin decreasein aqueous humor formation which was not statistically different from the control value. 4. Discussion In previously reported studies utilizing enucleated, arterially perfused cat eye (Macri and Brown, 1961), acetazolamide had been demonstrated to produce vasoconstriction in the anterior segment. Acetazolamide has also been demonstrated to cause a decreasein aqueous humor formation under conditions in which the aqueous humor formation rate had been stimulated by the use of acetylcholine (A&) plus eserine (Es) (Macri and Cevario, 1975). Hexamethonium was found to be an effective blocker of the inhibitory action of acetazo!nmide on this aqueoushumor production (Macri and Cevario, 1975). The aqueous humor production which was inhibited by acetazolamide in these earlier experiments is formed by an ultrafiltrative process (Macri, Cevario and Ballintine, 1974) probably induced by constriction of efferent ciliary process blood vessels (Macri and Cevario, 1973). It is possible that the epithelium of the ciliary processeshas a very high secretory capacity but is under-utilized due to insufficient substrate (ultrafiltrate from capillaries). As discussed in an earlier publication
126
F.
J.
MAC!RI
ASD
S. J.
C‘E\‘ARIO
group of experiments. acctazolamide and it,s antagonist, phencyclidine, were administ(ered together after basal value of aqueous production were determined. Aqueous humor production was then stimulated with Ach+Es. It was reasoned that if phencyclidinc were not blocking the action of acetazolamide, then the rise in aqueous humor production induced by Ach +Es should be of a magnitude less than that obtained in the first series of experiments where only Ach +Es was administered. As may be noted from the tables. the rate of aqueous humor formation was actually greater (4447 $/miii. vs. 27.31 ~l/niin). The blocking potential of phencyclidine was ~~~11demonstratetl in the final stc.1’ of this experiment. Removal of phencyclidine from t,he perfl~satc nledia caused the inflow rate to decrease immediately front 41.47 $/min to 21-60pljmin. The findings that phenc.\;clidinc can inhibit both the vascular and aqueous humor actions of acetazola,niide are strongly supportive of the view, jjresentrtl earlier. acctazolaniide lowers aqueoushumor formation by a primary vascular mechanism. If our vie\\-s on thy mechanism of acetazolamidc action on aqueoushumor pru(luction arc correct for the enuclea,ted eye, then experiments utilizing phencyclidinc, in living monkeys. should shedlight on the usefulnessof the enucleated eye as a physiologic model for studies of this tyl)r. as well as to indicat,e whether a vascular Inechanisni for the action of acctazolair~iclcapplies to the primate. Two seriesof oxpcrimcnts were performed on the eyes of living monkeys. In one .qtaritsof cxperimc~nts, anesthesia was maintained with urethan and in the sfacond s;cricsanesthesia was maintained II? phencpclidine. As previously reported ((‘evario and Nacri, 1974) ancst.hesiamaint,enancewit.h either tlrug, in a seriesof 30 monkeys, tlemonstrat~cdno difference in the control values for aqueous humor formation. In the current study, the control rate of aqueousproduct’ion for each group was almost itientical (3.09 &inin vs. 3.01 pljiiiin). dcet~azolamitle in a concentration of 50- 100 lrlgikg (administered i.v.) caused a 5390 decreasein the formation rate of aqueous humor in the urethan ancsthet,izrd monkeys. In the second seriesof experiments. it was tlecided to make the test of phencyclidinc’s ability to block acetazolamidc action in the monkey luort stringent : therefore, the dose of acetazolamide was increased to -?OO-300 mg/kg. ds may IW seen from Ta,ble IV, 1)heneyclidine almost completely inhibited the action of this 11mc11 higher concentration of acetazolamide on aclueous humor formation. The results obtained in lnonkeys for the inhibition of acetazolamide action by phencyclidine are therefore. in agreement with t,he similar findings obtained in the arterially perfusedZenucleated cat eye. 1Ye would suggestthat the data presented in these studies. is strong evidence that the primate eye in situ behavesas the enucleatetl cat eye and that the pharmacologic mechanismsproposed for acetazolamitlr a&on in the cat eye is very- probably alq~lical~leto the primate. tht
REFERESC’ES (‘evario, S. J. and 3Iacri. F. tJ. (1974). The inhibit,ory effect of pentobarbital Sa on ay~~~~ous J~un~or formation. Invest. Ophthalmol. 13, 384-K (‘hen. G., Ensor. C. K.. Kussel, D. and Bohner. B. (1959). The pharmacology of l-(l-~~l~rr~~i~~~clo~ hexyi) piperidine-HCl. J. Pharnl. h’xp. Themp. 127, ‘741-N. Domino, E. F. (1964). Neurobiology of phencyrlidine (Sernyl)-a drug xvith an unusual spectrum of pharmacological activity. Id. Rev. ?JeurobioZ. 6, 303. Mncri, F. J. and Brown, J. G. (1961). The constrictive actionof acetazolamide on the iris arteries of the cat. Arch. Ophthdmol. 66, 570-7.
I;r;HIHLTION
OF
~4(‘ETAZOLA3’IIDE
KY
PHEiTCYCLIDISE
1%
(Macri and (‘tlvario. 1975) the increased ultrafiltrate should be easily moved across the membrane if on? assumes this condition to be met. Therefore, it would have to be postulated that a,cetazolamide decreases the secretory capacity of the ciliary epithelium to inhibit the rate of aqueous humor formation. The question arises as to whether secretory inhibition by acetazolamide is a viable option in view of the findings that acetazolamidc action can be blocked by (J-6. It cannot be stated with any high degree of certainty, that C-6 does not relieve the inhibitory potential of, acetazolamide on the ciliary epithelium. The ability of C-6 to interfere with a,cetazolamide inhibition of carbonic auhydrase has yet to bc determined. A number of other pharmacologic agents hare been reported to decrease the rate of a~clurous hru~~or formation in the isolated eve preparation used her?. Among these are I,-el~inephrine. L-norepinephrinc. n.L-isoprotcrenol (Macri and C!evario, 1974a) pilocarpine, (Nacri ant1 Cevario, 1974b) the ganglionic stimulant DMPP (dimethylpllcti~lil)era,ziil~uiii). ouabain and acetazolamide (Jiacri and c(evari0, 1975). Although activity. these compou~itls arc from groups with basic diverse pharmacologic they all have> one other action in common which is to produce a vasoconstriction in the anterior segment of the eye. It has been postulated that these agents act upon intraoculn,r nicotinic (E-l) ganglion-like receptor sites, since in all cases, their vasocoll;;trictor activity is abolished by C-6. All of the above pharmacologic agents, which ha\~c I!clen found to reduce the rate of aqueous humor pro&uction in the cat whole eye prcpa8ration 11al.c I)t:en found to have this action also ljlccked by the administration of (‘-6. The association of’ rasoeonstriction and inhiijition of aqueous humor production by each of these agonists and the common response of C-6 to Hock each form of a&\-it!- leatl KS to suggest that the decrease in xqueoas humor formation produced by these agents is mediated by constriction of afferent ciliary process l~lood vessels to affect a decrease in ultrafiltration (Macri and C’cvario, 1974a; Macri and Cevario. 1974h ; Jlacri and C’evario, 1975). The primary locus of action of these drugs which produce the vasoconstriction is on, or proximal to intraocular neurogenic sites which have properties similar to nicotinic (E-l) sites of sympathetic ganglia. It could 1~ inferred that the mechanism of action proposed for the cat eye is also operative in the primate eye. To support this proposition a~ the findings reported here with phcncyclid in t-. Ye have shot~u it, the current study that. l~l~t~ncyclidinc is capble of blocking the vasoconstrictor action of acetazolam&le on the> isolated cat iris-ciliary body preparation. Since phencyclitlinc has been reljortctl to have no r/.-atlrenergic blocking properties (l~oiiiino, 1964) ; it would appear that the blockade of acetazolamide vasoconstriction I)y phencyclidine is at some site other than cc-atlrcnergic receptors. The magnitude of the decrease in perfusate flow observed in the isolated iris-ciliary body preparation by acetazolamide ma,s 3.12’!,. By itself, this value is of little quantitative significance other than to show that vascconstriction occurs. In the first of two sericts of experiments on arterially perfused enucleatetl eyes in which the rate of aqueous humor formation had been increased by Ach+Es, the subsequent adu ministration of acetazolarnide and phencyclidine induced no change in the rate of aqueous hnmor production. It hat1 I~en previously reported (Macri and Cevario: 1975) that, acetazolamide administered alone under similar conditions was capahlc of decreasing the inflow rate of aqueous humor lay 35%. A seco~ltl series of experiments were then performed on the whole eye preparation to test t’he validit#y of the blocking action of phencyclidine noted above. In this
INHIBITION
OF
Xacri,
ACETAZOLAMIDE
BY
PHENCYCLIDINE
IPi
F. J. and Cevario, 6. J. (1973). The induction of aqueous humor formation by the use of r2ch+eserine. Invest. Ophthnlmol. 12, 910-16. Xacri. F. J. and Cevario, S. J. (1974a). A pharmacodynamic study of the inhibit.ory effects of I,-norepinephrine, r.-epinephrine, and n.L-isoproterenol on aqueous humor formation in the enucleat,ed arterially perfused cat eye. Inzwt. Ophthdmol. 13, 39”~3. Jlacri. F. J. and Cevario. S. J. (1974)b. The dual nature of pilocarpine to stimulate or inhibit tlw formation of aqueous humor. Invest. Ophthnlmol. 13, 617-g. Mawi. 1~‘. .J.. Cevario. S. J. and Ballintine. E. J. (1974). The arterial pressure dependency of the aqueous humor format,ion induced by Ach rmeserine. Z/rw.sf. Ophthnlmol. 13, 1535. Mauri. F. J. and (‘evario, S. J. (1975). h possible vascular mechanism for the inhibition of aqueous humor formation by ouabain and acetazolamide. E.rZ). E!Ze Kex. 20, 56:1&l). .\Iacri. F. J., Dixon. R. L. and Rall. D. P. (1965). Aqueous humor turnover rates in the wt. I. Effect of acetazolamide. Iwest. Ophthdn/ol. 4, WT. l\lacri. P. .J. and O’Rourke. J. (1965). Measurements of aqueous Iiumor turnover rates in the cat. 1 I. Effect of acetazolamicle. Z~rwt. Ophthnlmol. 5, 927-34. .\Iacri. F. ,J. and O’Rourke. J. (1970). ?tIeasurements of aqueous humor tiunorer rates using a gamma probe>. Arch. Ophthdmd. 83, 711. Oppelt, IV. W. (1967). Measurement of aqueous humor formation rates by posterior-anterior ~~bamher perfusion with inulin: normal I-ahw and the effec? of carhonk anhydmse inhibition. III wst.
O~hthrrl~~~ol.
6, i&$7.
Blockade of the Ocular Effects of Acetazolamide by Phencyclidine
Phenryclidine. in low concentrations, has been found to block the vasoconstrictor action of acetazolamidc in t,he isolated, arterially perfused iris-ciliary body of the cat. Similar conwntrations of phenayclidine, in enwleated, arterially perfused whole cat eyes was found to prevent the action of acetazolamide to decrease the rate of aqueous humor tbrmat’ion. Esperiments in living rhesus monkeys also demonstrate that the inhibitory effects of acetnzolamide on aqueous humor formation cnn be effectively blocked by phencyclidiw. phrnryrlidine: vasoconstriction: t.at : Key words: aqueous humor: acetazolamide: monkey.
1. Introduction dcetazolamide has been demonstrated to produce vasoconstriction (Macri and Brown 1961; Macri and Cevario, 1975) and to decreasethe rate of aqueoushumor formation in the intact (Macri, Dixon and Rall, 1965; Oppelt, 1967) and cnucleated (Macri and (‘evario, 1975), arterially perfused cat eye. In the enucleated eye both of these actions are capable of being inhibited by the ganglionic blocking agent?hexamethonium (C-6) Macri and Ccvario, 1975). Blockage of acetazolamide action. hy hexamethonium has strengthened the concept that the action of acetazolamide is mediated by a local vascular constriction rather than by inhibition of carbonic anhydrase (Nacri acd Cevario: 19i5). It would be tlesiral)le to determine if a compound such as C-6, which has no known action on carbonic anhydrase, is also capable of blocking the action of acetazolamide on the eye of the anesthetized animal. Unfortunately, (~‘-6.is not amenable for this type stutly since its ganglionic blocking properties produce a marked decline of Moot I pressure which alters steady-state conditions of comparison. Fortuitously, another substance, phencpclidine, has been found to exhibit actions on the eye much like those of (‘-6. In the studies to be reported here. phencyclidine is shown to l)e a specific blocker of both the acetazolamide vascular and aqueous hunior activity in the cnucleated eye preparations. Phencyclidine is also demcnstrated to inhibit the actions of acetazolamide on aclueoushumor formation in the living rhesus monkey. 2. Methods Youug adult cats of either sex were utilized in this study. The armnalswere auesthetized with pentobarbital Na (30.0 mg/kg administered iutraperitoneallv). Ascorbic acid (‘LO.0 mg) was administeredin salinesolution, i.v. 5 min before the animal was killed and the eyes enucleated. The proceduresfor the preparation of the isolated, arterially perfusediris-ciliary body
and the whole cat eyes were identical to those previously reported (Macri and Brown, 1961; Macri and Cevario, 1973). Briefly, both preparations were perfusedintra-arterially with warmed (36°C) Eagles Basal Medium (EBM) at a pH of 7.4. The EBM solution was modified to contain 0.8% NaCl and 0.1 mg% ascorbic acid. No serum was used in the
F.
122
J.
MAC’XI
AA-I)
$.
.J. (‘I~V.-\IiIU
medium. Perfusate flow was measured utilizing a differential pressure-type tlowmeter (Macri and Brown, 1961). dqueous humor turnover in the enucleated, arterially perfusetl eyes \\eas determined by the method previously ut,ilized in the in viva cat (Jlacri il11cl O’Rourke, 1970). Male and female Mcccaca mzclattu (Rhesus) monkeys weighing between 2.0 and 3.5 kg were anesthetized with Sernylan (phencytlidine hydrochloride, 0.5 mg/kg). Small suhsequent doses of phencyclidine were given, when necessary, to maintain anesthesia. In a second series of experiments, Urethan (0.5 g/kg) was injected i.p. to maintain anesthesia, following the initial anesthetizing dose of phencyclidine. One femoral vein was cannulated in all monkeys for the administration of acetazolamide. The aqueous humor turnover in the living monkev was determined by the [14C]inulin dilution technique (Macri, Dixon and Rall, 1965)mo&fied asreported (Cevario and JIacri, 1974). Concentrations of drugs are expressed as sa1t.s. 3. Results Effects ia the iris-ciliury body preparation In seven experiments, phencyclidine (O-01 pg/ml) was perfused together with acetazolamide (20.0 pg/ml) through one long posterior ciliary artery of this preparation. Under this condition, vasoconstriction was not observed. Subsequent perfusion of the same preparations with acetazolamide alone, in all experiments, produced a decreasein arterial perfusion flow rate which averaged 3.12% (Table I).
Phencyclidine blockadeof acetazolamide vasoconstrictionin the isolated iris-cilia.ry body preparation
Control
Arterial perfusate Phenoyclidinef PO1 f&4
flow rate (~l/min) Acetazolamide (20.0 pg/ml)
*Meanfs.E.-seven preparations. tStatistically significant P < O-01 (paired
Acetazolamide WfJ I*@4
t-test).
Eflects on enucleated,arterially perfused cat eyes Six eyes were studied in this series(Table II). The rate of aqueoushumor formation was increased by the intra-arterial perfusion of Ach (O-01 rig/ml) +eserine (Es) (10.0 pg/ml). The formation rate of aqueous humor was found to increase from a basal level of 11.11. pl/min to 2’7.31 pl/ min. The addition of phencyclidine (0.01 pg/ml) to the perfusate media produced within 30 min a statistically significant, but small (3.7 &nin)
decrease in the aqueous
humor
inflow
rate. The addition
of acetazol-
amide (20.0 rug/ml) to the perfusion medium did not alter the rate of aqueous humor formation from those levels observed with phencyclidine alone. A previous report (Macri and Cevario, 1975) has demonstrated that the administration of acetazolamide to the Ach +Es stimulated eye, produced a 35% decreaseof the stimulated inflow rate. In a secondseriesof seven experiments (Table III), a different protocol was followed
INHIBITIOB
OF
ACETAZOLAMIDE
BY
123
PHEKC:TCLIDIXE
to verify the finding that phencyclidine could block the action of acetazolamide on aqueous humor production. After obtaining steady-state values of basal aqueous humor formation: the combination of aceta~olamide (20.0 mg/l) and phencyclidine
Block by phencyclidine of ncetazolamide inhibition of stinrulated aqueous humor form&ion in ellucleated arterially perfused cat eye (Series 1)
cbntro1
Il.11 (6) 2 141
Achl
l&wine
Inflow (rljmin) Ach +Eserine + Phencwlidinr
27.41 (s) *2.7&4
dch + Eserine 7 Phencyclidine + acetazolamide
23.63 (8) *7.2-l
-wean*S.E. Number of eyes shown in brackets. (R) Statistically significant P < 0.05 paired l-test. (n.s.) Not statistically significant. Ach 0.01 np/ml+eserine 10.0 pg/ml: phencyclidine
23.61 (n.8.) f%lX
O+l
&ml:
scetazolamide
20.0 pg/ml.
(0.01 pg/ml) were perfused first. No significant effect on basal levels of aqueous humor formation was noted. Subsequent arterial perfusion of the above drugs plus the stimulants Sch (0.01 rig/ml) and Es (10.0 pg/ml) produced a statistically significant, increase in the rate of aqueous humor formed (17.23 pl/min to 44.47 pl/min). TABLE
III
Block by phencyclidine of ncetazolnmide inhibition of stimulated rrqueow humorformation in enucleated arterially perfused cat eye (Series 2) Inflow
(‘ontrol 17,65(7) :t l%J
Acetazolamide +phencyclidine 17.23 (n.5.) j1.70
(pljmin) Acetazolamide +phencyclidine +Ach+eserine 44.47 +5~83
Mean&s.& Kumber of eyes shown in brackets. (8) Statistically significant P < 0.05, paired f-test. (n.s.) Not statistically significant. Ach 0.01 ng/ml+eserine 10.0 pz/mi: phencyclidine
(s)
0.01 &ml;
Acetazolamidcf Achfrswine 21.60
(s)
*“.jz
acctazolamide
204 pa/ml.
Alternating the arterial perfusate to another solution which did not contain the blocking agent phencyclidine, but had identical concentrations of Ach, Es and acetazolamide caused a 51% reduction in the rate of aqueous humor formation (from 44.47 @nin to 21.60 &‘min). E$ects on aqueoushumor production
i,n
anesthetizedrhesus,monkeys
A previous study of the rhesusmonkey (Cevario and Macri, 1974), hasdemonstrated that the rate of aqueous humor formation remains identical when anesthesia is induced by phencyclidine and maintained with either phencyclidine or urethan.
124
F. ,J. MAC’RI
ASI)
F. 5. (.‘EVAHIO
Ten experinwnts were pcrformetl on rhesus monkeys in which the ancst,hesial was maintained with w&an after incluction with phencyclidine (Table I\?). At the rw.l of 1 hr. at which time the eflects of l~henc~clidinr wrct presumably tlissipatetl ((‘hen, Xnsor, Ruswl ancl Rohner. 1959). tllp rate of i~ql~eoushw~lor fortl&on \vas clrtermined in one ttye for one how tinir lwriod. The average valw 0l)taitrctl for this il,
Actdo>lsof ncetctzolawide
on aqurous
hwror
fovwaatio~~ i,n the desus monkey
control interval was 3.09 pl/min. Acetazolamidc (50-100 mg/kg i-v.) was then administered to each monkey and following a 30 min equilibration period, aqueous humor production was determined for a 1 hr interval in the second eye. Acetazolamide was observed to decreaset)hc mtc of aqueoushumor formation by 1.64 pljniin to a value of 145 pl/min. In a second seriesof 10 rhesus monkeysi anesthesia was induced and maintained with phencyclidine alone. The sameprotocol was followed as above except that the dose of acctazolamide was increawtl to 200&300 nlg/kg. Control values of aqueous humor formation averaged 3.01 $jmin, which was not statistically different from control values of the urethane supplemented animals. Acetazolamide administration causeda 0.42 ~ljmin decreasein aqueous humor formation which was not statistically different from the control value. 4. Discussion In previously reported studies utilizing enucleated, arterially perfused cat eye (Macri and Brown, 1961), acetazolamide had been demonstrated to produce vasoconstriction in the anterior segment. Acetazolamide has also been demonstrated to cause a decreasein aqueous humor formation under conditions in which the aqueous humor formation rate had been stimulated by the use of acetylcholine (A&) plus eserine (Es) (Macri and Cevario, 1975). Hexamethonium was found to be an effective blocker of the inhibitory action of acetazo!nmide on this aqueoushumor production (Macri and Cevario, 1975). The aqueous humor production which was inhibited by acetazolamide in these earlier experiments is formed by an ultrafiltrative process (Macri, Cevario and Ballintine, 1974) probably induced by constriction of efferent ciliary process blood vessels (Macri and Cevario, 1973). It is possible that the epithelium of the ciliary processeshas a very high secretory capacity but is under-utilized due to insufficient substrate (ultrafiltrate from capillaries). As discussed in an earlier publication
126
F.
J.
MAC!RI
ASD
S. J.
C‘E\‘ARIO
group of experiments. acctazolamide and it,s antagonist, phencyclidine, were administ(ered together after basal value of aqueous production were determined. Aqueous humor production was then stimulated with Ach+Es. It was reasoned that if phencyclidinc were not blocking the action of acetazolamide, then the rise in aqueous humor production induced by Ach +Es should be of a magnitude less than that obtained in the first series of experiments where only Ach +Es was administered. As may be noted from the tables. the rate of aqueous humor formation was actually greater (4447 $/miii. vs. 27.31 ~l/niin). The blocking potential of phencyclidine was ~~~11demonstratetl in the final stc.1’ of this experiment. Removal of phencyclidine from t,he perfl~satc nledia caused the inflow rate to decrease immediately front 41.47 $/min to 21-60pljmin. The findings that phenc.\;clidinc can inhibit both the vascular and aqueous humor actions of acetazola,niide are strongly supportive of the view, jjresentrtl earlier. acctazolaniide lowers aqueoushumor formation by a primary vascular mechanism. If our vie\\-s on thy mechanism of acetazolamidc action on aqueoushumor pru(luction arc correct for the enuclea,ted eye, then experiments utilizing phencyclidinc, in living monkeys. should shedlight on the usefulnessof the enucleated eye as a physiologic model for studies of this tyl)r. as well as to indicat,e whether a vascular Inechanisni for the action of acctazolair~iclcapplies to the primate. Two seriesof oxpcrimcnts were performed on the eyes of living monkeys. In one .qtaritsof cxperimc~nts, anesthesia was maintained with urethan and in the sfacond s;cricsanesthesia was maintained II? phencpclidine. As previously reported ((‘evario and Nacri, 1974) ancst.hesiamaint,enancewit.h either tlrug, in a seriesof 30 monkeys, tlemonstrat~cdno difference in the control values for aqueous humor formation. In the current study, the control rate of aqueousproduct’ion for each group was almost itientical (3.09 &inin vs. 3.01 pljiiiin). dcet~azolamitle in a concentration of 50- 100 lrlgikg (administered i.v.) caused a 5390 decreasein the formation rate of aqueous humor in the urethan ancsthet,izrd monkeys. In the second seriesof experiments. it was tlecided to make the test of phencyclidinc’s ability to block acetazolamidc action in the monkey luort stringent : therefore, the dose of acetazolamide was increased to -?OO-300 mg/kg. ds may IW seen from Ta,ble IV, 1)heneyclidine almost completely inhibited the action of this 11mc11 higher concentration of acetazolamide on aclueous humor formation. The results obtained in lnonkeys for the inhibition of acetazolamide action by phencyclidine are therefore. in agreement with t,he similar findings obtained in the arterially perfusedZenucleated cat eye. 1Ye would suggestthat the data presented in these studies. is strong evidence that the primate eye in situ behavesas the enucleatetl cat eye and that the pharmacologic mechanismsproposed for acetazolamitlr a&on in the cat eye is very- probably alq~lical~leto the primate. tht
REFERESC’ES (‘evario, S. J. and 3Iacri. F. tJ. (1974). The inhibit,ory effect of pentobarbital Sa on ay~~~~ous J~un~or formation. Invest. Ophthalmol. 13, 384-K (‘hen. G., Ensor. C. K.. Kussel, D. and Bohner. B. (1959). The pharmacology of l-(l-~~l~rr~~i~~~clo~ hexyi) piperidine-HCl. J. Pharnl. h’xp. Themp. 127, ‘741-N. Domino, E. F. (1964). Neurobiology of phencyrlidine (Sernyl)-a drug xvith an unusual spectrum of pharmacological activity. Id. Rev. ?JeurobioZ. 6, 303. Mncri, F. J. and Brown, J. G. (1961). The constrictive actionof acetazolamide on the iris arteries of the cat. Arch. Ophthdmol. 66, 570-7.
I;r;HIHLTION
OF
~4(‘ETAZOLA3’IIDE
KY
PHEiTCYCLIDISE
1%
(Macri and (‘tlvario. 1975) the increased ultrafiltrate should be easily moved across the membrane if on? assumes this condition to be met. Therefore, it would have to be postulated that a,cetazolamide decreases the secretory capacity of the ciliary epithelium to inhibit the rate of aqueous humor formation. The question arises as to whether secretory inhibition by acetazolamide is a viable option in view of the findings that acetazolamidc action can be blocked by (J-6. It cannot be stated with any high degree of certainty, that C-6 does not relieve the inhibitory potential of, acetazolamide on the ciliary epithelium. The ability of C-6 to interfere with a,cetazolamide inhibition of carbonic auhydrase has yet to bc determined. A number of other pharmacologic agents hare been reported to decrease the rate of a~clurous hru~~or formation in the isolated eve preparation used her?. Among these are I,-el~inephrine. L-norepinephrinc. n.L-isoprotcrenol (Macri and C!evario, 1974a) pilocarpine, (Nacri ant1 Cevario, 1974b) the ganglionic stimulant DMPP (dimethylpllcti~lil)era,ziil~uiii). ouabain and acetazolamide (Jiacri and c(evari0, 1975). Although activity. these compou~itls arc from groups with basic diverse pharmacologic they all have> one other action in common which is to produce a vasoconstriction in the anterior segment of the eye. It has been postulated that these agents act upon intraoculn,r nicotinic (E-l) ganglion-like receptor sites, since in all cases, their vasocoll;;trictor activity is abolished by C-6. All of the above pharmacologic agents, which ha\~c I!clen found to reduce the rate of aqueous humor pro&uction in the cat whole eye prcpa8ration 11al.c I)t:en found to have this action also ljlccked by the administration of (‘-6. The association of’ rasoeonstriction and inhiijition of aqueous humor production by each of these agonists and the common response of C-6 to Hock each form of a&\-it!- leatl KS to suggest that the decrease in xqueoas humor formation produced by these agents is mediated by constriction of afferent ciliary process l~lood vessels to affect a decrease in ultrafiltration (Macri and C’cvario, 1974a; Macri and Cevario. 1974h ; Jlacri and C’evario, 1975). The primary locus of action of these drugs which produce the vasoconstriction is on, or proximal to intraocular neurogenic sites which have properties similar to nicotinic (E-l) sites of sympathetic ganglia. It could 1~ inferred that the mechanism of action proposed for the cat eye is also operative in the primate eye. To support this proposition a~ the findings reported here with phcncyclid in t-. Ye have shot~u it, the current study that. l~l~t~ncyclidinc is capble of blocking the vasoconstrictor action of acetazolam&le on the> isolated cat iris-ciliary body preparation. Since phencyclitlinc has been reljortctl to have no r/.-atlrenergic blocking properties (l~oiiiino, 1964) ; it would appear that the blockade of acetazolamide vasoconstriction I)y phencyclidine is at some site other than cc-atlrcnergic receptors. The magnitude of the decrease in perfusate flow observed in the isolated iris-ciliary body preparation by acetazolamide ma,s 3.12’!,. By itself, this value is of little quantitative significance other than to show that vascconstriction occurs. In the first of two sericts of experiments on arterially perfused enucleatetl eyes in which the rate of aqueous humor formation had been increased by Ach+Es, the subsequent adu ministration of acetazolarnide and phencyclidine induced no change in the rate of aqueous hnmor production. It hat1 I~en previously reported (Macri and Cevario: 1975) that, acetazolamide administered alone under similar conditions was capahlc of decreasing the inflow rate of aqueous humor lay 35%. A seco~ltl series of experiments were then performed on the whole eye preparation to test t’he validit#y of the blocking action of phencyclidine noted above. In this
INHIBITION
OF
Xacri,
ACETAZOLAMIDE
BY
PHENCYCLIDINE
IPi
F. J. and Cevario, 6. J. (1973). The induction of aqueous humor formation by the use of r2ch+eserine. Invest. Ophthnlmol. 12, 910-16. Xacri. F. J. and Cevario, S. J. (1974a). A pharmacodynamic study of the inhibit.ory effects of I,-norepinephrine, r.-epinephrine, and n.L-isoproterenol on aqueous humor formation in the enucleat,ed arterially perfused cat eye. Inzwt. Ophthdmol. 13, 39”~3. Jlacri. F. J. and Cevario. S. J. (1974)b. The dual nature of pilocarpine to stimulate or inhibit tlw formation of aqueous humor. Invest. Ophthnlmol. 13, 617-g. Mawi. 1~‘. .J.. Cevario. S. J. and Ballintine. E. J. (1974). The arterial pressure dependency of the aqueous humor format,ion induced by Ach rmeserine. Z/rw.sf. Ophthnlmol. 13, 1535. Mauri. F. J. and (‘evario, S. J. (1975). h possible vascular mechanism for the inhibition of aqueous humor formation by ouabain and acetazolamide. E.rZ). E!Ze Kex. 20, 56:1&l). .\Iacri. F. J., Dixon. R. L. and Rall. D. P. (1965). Aqueous humor turnover rates in the wt. I. Effect of acetazolamide. Iwest. Ophthdn/ol. 4, WT. l\lacri. P. .J. and O’Rourke. J. (1965). Measurements of aqueous Iiumor turnover rates in the cat. 1 I. Effect of acetazolamicle. Z~rwt. Ophthnlmol. 5, 927-34. .\Iacri. F. ,J. and O’Rourke. J. (1970). ?tIeasurements of aqueous humor tiunorer rates using a gamma probe>. Arch. Ophthdmd. 83, 711. Oppelt, IV. W. (1967). Measurement of aqueous humor formation rates by posterior-anterior ~~bamher perfusion with inulin: normal I-ahw and the effec? of carhonk anhydmse inhibition. III wst.
O~hthrrl~~~ol.
6, i&$7.