Download Drug responses of adenylate cyclase in iris-ciliary body

396
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / March 1985
Vol. 26
Drug Responses of Adenylote Cyclose in Iris-Ciliory Body
Determined by Adenine Lobe/ling
Thomas W. Mirrag* and Anne Tormay
The intracellular adenine nucleotide pool of rabbit irisciliary body was labelled by uptake of 3H-adenine in vitro.
A variety of agents were tested for their ability to stimulate
or inhibit the incorporation of radioactivity into cyclic AMP
formed from ATP labelled with 3H-adenine. Isoproterenol,
vasoactive intestinal peptide, forskolin, and prostaglandin
E2 stimulated incorporation of label 3-10-fold in 15-20 min
compared with paired tissues not treated with hormone,
whereas histamine, serotonin, substance P, and bradykinin
were inactive. Clonidine, a-methylnorepinephrine, and dopa mine decreased the rate of incorporation of label into the
cyclic-AMP pool in tissues that showed high spontaneous
basal rates. In low-basal tissues these drugs were inactive
by themselves but clonidine and a-methylnorepinephrine
blocked the stimulation effected by isoproterenol. The findings indicate that several receptor-coupled adenylate cyclase
systems are present in ICB and that dual adrenergic control
of adenylate cyclase through positive and negative coupling
of adrenergic receptors probably occurs. The negatively
coupled adrenergic receptors appear to be similar to the a2subclass of adrenergic receptor described in other tissues.
These observations suggest a role for the large number of
a2-adrenergic-binding sites found in albino rabbit iris-ciliary
body by ligand binding assays. Invest Ophthalmol Vis Sci
26:396-399, 1985
Cyclic-AMP is thought to play a significant role in
mediating drug effects on intraocular pressure in the
mammalian eye because the ciliary processes have
high adenylate cyclase activity.1-2 Activation of adenylate cyclase, either through agonist occupancy of
receptors coupled to the enzyme, or at other levels
in the coupling between receptors and the enzyme,
leads to a rise in cyclic-AMP, and is associated with
a fall in intraocular pressure.3
Many hormones elevate the intracellular cyclicAMP, such as the /3-adrenergic effects of norepinephrine, prostaglandin E, histamine, and vasoactive intestinal peptide, to name a few. However, some
hormones have been shown to inhibit adenylate
cyclase activity in specific tissues, as for example, the
a-adrenergic effects of norepinephrine, opiates, and
the muscarinic effect of acetylcholine. The possibility
of a variety of adenylate cyclase coupled receptors
with both positive and negative coupling to the
adenylate cyclase in iris-ciliary body, initiated this
study.
Stimulatory hormone effects on ciliary body adenylate cyclase have been determined mainly by two
methods, either measurement of the rise in cyclicAMP in the aqueous humor following drug treatment
in vivo4 or by in vitro biochemical determination in
membranes prepared from ciliary processes.12'5 These
methods do not give good responses for negatively
coupled receptors that are more easily detected by
labelling of intracellular ATP in intact cells, to determine the flux of the radioactive label into the cyclicAMP pool. A relatively simple pulse method for
measuring the effects of hormones and other agents
on the formation of cyclic-AMP has been developed
for brain slices.6 In the present experiments, we have
adapted this technique to pieces of iris-ciliary body
(ICB) in vitro and determined stimulation or inhibition of adenylate cyclase by measuring the rate of
incorporation of radioactivity into cyclic-AMP formed
as a result of cyclization of 3H-adenylyl-labelled ATP.
Materials and Methods. Intracellular labelling of
ICB tissue: The ICB was dissected intact from albino
New Zealand rabbits (1.5-2 kg) killed by an overdose
of sodium pentobarbital or air embolism. The whole
tissue was incubated in 2 ml of continuously gassed
PK buffer (Krebs buffer with 10 mM pyruvate)
containing 3H-adenine (0.66 (xM). A linear uptake of
label over the period 10-60 min was found. Routine
intracellular labelling was done with 6 ICB tissues
incubated in 4 ml of PK buffer containing 0.2 mM
indomethacin, 5 X 10~5 M 8-phenyltheophylline, 0.66
[iM 3H-adenine (New England Nuclear Corp.; Boston,
MA, 20 Ci/mMole) for 20 min at 30°C. About 50%
of the total label was taken up by the tissues under
these conditions (approximately 20 nmoles adenine
per ICB).
Drug responses: After prelabelling with 3 H-adenine
the tissues were rinsed, quartered, and each piece put
into 0.5 ml of PK buffer containing the drug being
tested and 0.5 mM isobutyl methyl xanthine (phosphodiesterase inhibitor). Tissues were incubated for
15 or 20 min with surface gassing (95% O 2 , 5% CO2)
in a shaker bath at 30°C. The incubation was terminated by addition of 0.5 ml of 6% trichloroacetic acid
to the tissue and buffer.7 Cyclic-AMP was separated
from the acid extract by chromatography,8 and radioactivity associated with cyclic-AMP counted.
Quarter-tissues from one ICB were kept as a
matched set, with the basal (no drug) conversion of
label into cyclic-AMP determined on one or two of
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397
Reporrs
No. 3
LOW BASAL CYCLASE/EFFECT OF a 2 AGONISTS
BASAL CONVERSION OF
ATP TO c-AMP BY ICB TISSUES IN 15 MIN.
(6)
4.0
T
(6)
(6)
T
x
i 3.0
2.0
(6)
0.2-0.3
0.3-0.5 0.5-0.75
0.75-1.5 1.5-3.0
% CONVERSION TO c-AMP
Fig. 1. Distribution of the basal rates for percent of total
3
H-adenine uptake incorporated into the cyclic-AMP pool. ICB
tissues showed a 10-fold range of basal incorporation when incubated
for 15 min in buffer alone, necessitating that quartered tissues of
one ICB be maintained as a set for drug experiments.
the quarters, leaving two or three quarters of the
same set available for incubation with test drugs.
The percent conversion is calculated from the ratio
of radioactivity in the cyclic-AMP fraction to the
total radioactivity contained in the acid nucleotide
extract. For matched tissues, the effect of drug treatment is expressed as the stimulation index ± SEM,
which is the multiple of the basal conversion rate
(determined from the no-drug quarter tissue of the
same set) needed to equal the conversion rate in
presence of the drug.
Materials: Drugs and other buffer ingredients were
obtained from Sigma Chemical Co. (St. Louis, MO)
or from Cal Biochem (La Jolla, CA). The experiments
were performed in accordance with the ARVO Resolution on the Use of Animals in Research.
Table 1. Cyclic-AMP stimulation indices of biogenic
amines, peptides, and lipid derivatives
Dose
Biogenic amine
Isoproterenol, 1 juM
Serotonin, 10 /xM
Histamine, 10 nM
Dopamine, 10 ^M
Peptide
Vasoactive intestinal peptide,
0.1 »M
Substance P, 1 nM
Bradykinin, 1 ^M
Lipid derivative
Arachidonic acid, 1 nM
Forskolin, 10 fiM
PGE 2 ,0.1 fiM
Stimulation index
3.70
0.93
1.80
1.27
± 0.33 (n
± 0 . 1 5 (n
± 0.26 (n
±0.16(n
=
=
=
=
6)
6)
5)
7)
10.65 ± 2.17 (n = 6)
0.81 ± 0.17 (n = 3)
1.59 ± 0 . 1 2 ( n = 5)
0.90 ± 0.35 (n = 3)
3.30 ± 0.54 (n = 10)
5.14 ±0.58 (n = 5)
ISO.IO"6 ISO.IO"6 CLONJO"5
+
CLON.IO"5
ISO.IO"6 ISO.IO'6 aMNE,
+
io-5
aMNE.IO" 5
Fig. 2. The inhibitory effect of clonidine (CLON) and «-methyl
norepinephrine («-MNE) on the stimulation index of "low-basal"
tissues activated with 1 nM isoproterenol (ISO).
Results. The response to added drugs was determined by incubating 3H-adenine-prelabelled quarters
of ICB tissue for 15 or 20 min with supramaximal
concentrations of the test drug. The basal conversion
of intracellular label into cyclic-AMP (ie, with no
added drugs) was quite variable (Fig. 1). The majority
of ICBs showed basal conversion rates in the range
of 0.2-0.5%, which is similar to basal rates found in
other tissues.6 However, some tissues showed somewhat higher basal rates, and about 20% were in the
high range (0.75%-3%, Fig. 1). The majority of tissues
that had low basal activities gave good responses with
activators of adenylate cyclase in terms of the stimulation index. Since this quantity is denned as the
factor by which basal activity must be multiplied in
order to equal the activity in presence of the agent
being tested, stimulation indices will be numerically
larger for low-basal tissues than for high-basal ones.
The low-basal tissues were used to assess which
agents were activators of adenylate cyclase. Isoproterenol is an effective stimulant of cyclic-AMP turnover,
showing coupling of adenylate cyclase to (8-adrenergic
receptors, but other biogenic amines, which are activators of adenylate cyclase in brain are poor stimulators in rabbit ICB (Table 1). Three peptides to
which ICB is responsive also were tested but only
VIP is a cyclic-AMP activator. Among lipid derivatives
tested both forskolin and PGE2 show significant activity but arachidonic acid gave no response, as
expected, since conversion to PGs is prevented by
the presence of indomethacin in the incubation medium.
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INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / March 1985
398
HIGH BASAL CYCLASE / EFFECT O F a 2 AGONISTS
BASAL
Fig. 3. The inhibitory effect of clonidine (CLON), a-methyl
norepinephrine (a-MNE) and dopamine (DA) on the stimulation
index of "high-basal" tissues.
Tissues with low basal activities were tested for the
possible presence of a-adrenergic receptors negatively
coupled to adenylate cyclase with drugs having a
selectivity for the a2-receptor subclass. Both clonidine
and a-methylnorepinephrine neither stimulated nor
inhibited basal adenylate cyclase activity when given
alone to low-basal tissues (Fig. 2). However when the
cyclase activity of the tissue was stimulated with 1
nM isoproterenol, the inhibitory effects of the a2receptor agonists added together with the isoproterenol
became apparent. At this dose ratio of stimulant to
inhibitant (10~6 M:10~5 M), the inhibitory effect of
clonidine was not statistically significant but was
highly significant for a-methyl norepinephrine.
Tissues with a high basal activity also showed
a-adrenergic inhibition of adenylate cyclase activity.
In this case no additional stimulation by an exogenous
agent (isoproterenol) is required, and both clonidine
and a-methylnorepinephrine cause a significant reduction in activity when given alone (Fig. 3). Dopamine was also found to be inhibitory in high-basal
tissues, but this response could be due to its activity
at a2-adrenergic receptors or to attenuation of endogenous norepinephrine release by activity at presynaptic
autoinhibitory dopamine receptors.
Discussion. The increase of aqueous humor cyclicAMP following an activating stimulus probably reflects
the rise of cyclic-AMP in ciliary epithelial cells.
However, this in vivo method cannot determine
inhibitory influences since the fall of intracellular
cyclic-AMP is not paralleled by a decline of the basal
concentration of aqueous humor cyclic-AMP, which
is close to that of the plasma.
Intracellular labelling can be used as an indirect
alternative to other in vitro methods, such as determining the cyclic-AMP content of a tissue by analytic
methods (RIA analysis) or radiometric assay of ade-
Vol. 26
nylate cyclase in membrane preparations.2'5 In its
present simple form, only changes in radioactivity of
the cyclic-AMP fraction in response to drug treatments
is measured, and the absolute concentration of cyclicAMP is not determined. Thus, the stimulation indices
are not quantitative measurements, only relative
changes, and the coupling of receptors to adenylate
cyclase enzyme is determined indirectly. Nevertheless,
the present simple technique provides important preliminary information showing the absence or presence
of other cyclic-AMP activator receptor systems in the
rabbit ICB. All of the activators tested (Table 1) have
known effects on IOP, but these do not correlate with
their cyclic-AMP effects. Beta-adrenergic agonists (eg,
isoproterenol) lower IOP, as does forskolin, whereas
VIP and PGE2 raise IOP (PGE 2 can lower IOP at
some doses). The ocular hypertension reported to
occur with VIP and PGE 2 could be related to the
intraocular inflammatory response induced by these
agents, masking their hypotensive action.
A significant finding is that adenylate cyclase of
ICB is coupled to inhibitory receptors, which appear
to be a2-adrenergic receptors. Binding studies have
shown that this receptor subclass comprises nearly
half of the total adrenergic receptors in this tissue
and are almost twice as numerous as «|-adrenergic
receptors.9 Receptors of the a2-subtype are present
on adrenergic nerve terminals as presynaptic autoreceptors but also are found as postsynaptic receptors
in vascular tissues and negatively coupled to adenylate
cyclase in platelets10 and in adipocytes." This subtype
of adrenergic receptor also mediates drug effects on
fluid absorption and fluid secretion in gastrointestinal
epithelia12 and a2-receptor selective agonists are known
to lower IOP.13 The fact that clonidine, and a-methyl
norepinephrine, both agonists with a 2 -adrenoreceptor
selectivity, can block the isoproterenol stimulation of
adenylate cyclase is strong evidence for a dual adrenergic control system for adenylate cyclase in rabbit ICB.
Key words: iris ciliary body, adenylate cyclase, activators,
inhibitors, adrenergic receptors
From the Departments of Pharmacology and Ophthalmology,*
Mount Sinai School of Medicine, New York, New York. Supported
by research grant EY-02619 and Core Center Grant EY 01867
from the National Eye Institute, National Institutes of Health.
Submitted for publication: March 2, 1984. Reprint requests: Dr.
T. W. Mittag, Department of Pharmacology, Mount Sinai School
of Medicine, One Gustave L. Levy Place, New York, NY 10029.
References
1. Waitzman MB and Woods WD: Some characteristics of an
adenylate cyclase preparation from rabbit ciliary process tissue.
Exp Eye Res 12:99, 1971.
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No. 3
Reporrs
2. Nathanson JA: Adrenergic regulation of intraocular pressure:
Identification of beta2-adrenergic stimulated adenylate cyclase
in ciliary process epithelium. Proc Natl Acad Sci USA 77:
7421, 1981.
3. Rowland JM and Potter DE: Effects of adrenergic drugs on
aqueous c-AMP and c-GMP and intraocular pressure. Albrecht
Von Graefes Arch Klin Ophthalmol 212:65, 1979.
4. Neufeld AH, Jampol LM, and Sears MC: Cyclic-AMP in the
aqueous humor: The effects of adrenergic agents. Exp Eye Res
14:242, 1972.
5. Mittag TW and Tormay A: Desensitization of the 0-adrenergic
receptor and adenylate cyclase complex in rabbit iris-ciliary
body induced by topical epinephrine. Exp Eye Res 33:497,
1981.
6. Shimizu H, Daly JW, and Creveling CR: A radioisotopic
method for measuring the formation of adenosine 3'-5' cyclic
monophosphate in incubated brain slices. J Neurochem 16:
1619, 1969.
7. Ferrendelli JA, Rubin EH, Orr HT, Kinscherf DA, and Lowry
OH: Measurement of cyclic nucleotides in histologically defined
samples of brain and retina. Anal Biochem 78:252, 1977.
399
8. Salomon Y, Londos C, and Rodbell M: A highly sensitive
adenylate cyclase assay. Anal Biochem 58:541, 1974.
9. Mittag TW and Tormay A: Adrenergic receptor subtypes in
rabbit iris ciliary body membranes; radioligand binding studies.
Exp Eye Res, Vol. 40, 1985, in press.
10. Jakobs KH, Saur W, and Schultz G: Reduction of adenylate
cyclase in lysates of human platelets by the a-adrenergic
component of epinephrine. J Cyclic Nucleotide Res 2:381,
1981.
11. Bylund DB and U'Prichard DC: Characterization of «i and
«2-adrenergic receptors. Int Rev Neurobiol 24:343, 1983.
12. Nakaki T, Nakadate T, Yamamoto S, and Kato R: «-2
adrenergic inhibition of intestinal secretion induced by prostaglandin Ei, vasoactive intestinal peptide and dibutyryl cyclic
AMP in the rat jejunum. J Pharmacol Exp Ther 221:637,
1982.
13. Innemee HC, de Jonje A, van Meel JCA, Timmermans
PBMWM, and van Zwieten PA: The effect of selective a,- and
a2-adrenoreceptor stimulation on intraocular pressure in the
conscious rabbit. Naunyn Schmiedeberg's Arch Pharmacol
316:294, 1982.
Cotecholomines in Humon Aqueous Humor
Graham E. Trope* and Alan G. Rumleyf
Aqueous humor catecholamine levels were measured in 14
patients admitted to hospital for cataract or glaucoma
surgery. Norepinephrine was detected in all patients. Epinephrine was detected in one patient who had received a
preoperative retrobulbar injection of epinephrine. Dopamine
was not detected in any patients. The highest level of
norepinephrine was detected in cataract patients with normal
intraocular pressures (mean - 7.18 nmol/1). Invest
Ophthalmol Vis Sci 26:399-401, 1985
It is presently believed by some workers that
aqueous humor does not contain catecholamines. It
has been suggested that the mechanisms of inactivation of released catecholamines are so efficient that it
is not possible to detect norepinephrine in aqueous
humor.1"3 Recently sensitive radioenzymatic techniques have been developed, which allow for the
detection of catecholamines present in minute concentration in various fluids. Catecholamines have
been reported to exist in plasma,4 and we have
detected them in as little as 10-20 /A of tears.
The main purpose of this study was to confirm the
presence of catecholamines in human aqueous humor
and to extend the study to determine catecholamine
levels in patients with glaucoma.
Materials and Methods. Fourteen aqueous humor
samples from 14 patients were studied. Thirteen
patients were admitted for routine eye surgery under
general anaesthetic, one patient was admitted for
surgery under local anesthetic. Aqueous humor samples were obtained from three groups of patients.
Group 1: This group consisted of six patients
admitted for uniocular intracapsular lens extraction
(cases 1-6) under general anaesthetic. None received
preoperative sympathetic eyedrops. All underwent a
limbal paracentesis with removal of aqueous humor
on the operating table before the anterior chamber
was entered with surgical instruments. Once aqueous
humor had been withdrawn into a tuberculin syringe,
sympathetic drops were applied to the eye to dilate
the pupil for further surgery.
Group 2: This group consisted of four patients
newly diagnosed as having open-angle glaucoma
(OAG). These patients were involved in a study to
determine the role of primary trabeculectomy in
patients with this disease5 (cases 7-10). None of these
patients had ever received topical or systemic antiglaucoma therapy. Three of the patients had primary
open-angle glaucoma (POAG). One had pigmentary
glaucoma (case 8). Three of the patients underwent
trabeculectomy under general anesthesia (cases 7-9).
One patient, however, had a local anesthetic. In this
case a retrobulbar injection of 1% lignocaine plus 1:
100,000 adrenaline was used (case 10). Samples of
aqueous humor were removed through a limbal
puncture under the superficial trabeculectomy flap
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