2-Adrenergic-Stimulated Adenylate Cyclase in Ciliary Process Epithelium* (Cyclic AMP/Receptors/Secretion/Glaucoma) JAMES A

Proc. Natl. Acad. Sci. USA Vol. 77, No. 12, pp. 7420-7424, December 1980 Medical Sciences

Adrenergic regulation of intraocular pressure: Identification of #2-adrenergic-stimulated adenylate cyclase in ciliary process epithelium* (cyclic AMP/receptors/secretion/glaucoma) JAMES A. NATHANSON Departments of Neurology and Pharmacology, Harvard Medical School, Massachusetts General Hospital, Boston, Massachusetts 02114 Communicated by Paul Greengard, August 18, 1980

ABSTRACT To determine the site and possible mechanism adrenergic receptors in the ciliary processes separate from those of action of adrenergic agents in regulating intraocular pressure, in the iris and ciliary body; (ii) whether these receptors are experiments were undertaken to identify, localize, and char- subtype or /2; and (iii) whether the receptors are enriched acterize ft-adrenergic receptors associated with adenylate cy- f3, clase in various ocular tissues involved in secretion and reab- in the ciliary process epithelial cells or in the ciliary vascula- sorption of aqueous humor. The ciliary process epithelium was ture. found to be enriched in an adenylate cyclase with pharmaco- METHODS logical characteristics indicative of a predominance of 2- adrenergic receptors. The results are consistent with the possi- The methods for the present experiments were based on evi- bility that the adrenergic nervous system may regulate aqueous dence that indicates that receptor binding of fl-adrenergic humor production through a direct effect on secretion. The data agonists is associated with a concomitant activation of the en- also are relevant to the potential development of drugs that can zyme adenylate cyclase which synthesizes cyclic AMP from control increased intraocular pressure. ATP (7, 8). Data from a number of different tissues indicate that the characteristics of binding of 0-adrenergic agonists and Open-angle glaucoma is a progressive disorder of increased antagonists to the 3-adrenergic receptor are quite similar to the intraocular pressure (lOP) which results from excess accumu- ability of these same compounds either to activate adenylate lation of aqueous humor, a fluid that is formed by the ciliary cyclase or to block the stimulation of the enzyme by f3-adren- processes in the posterior ocular chamber and normally leaves ergic agonists (9). the eye through the trabecular meshwork and Schlemm's canal, Tissue Dissection. Most experiments were performed with located in the lateral angle of the anterior chamber. eyes enucleated from male New Zealand White rabbits (2-3 Considerable evidence suggests that the adrenergic nervous kg) that had been anesthetized and killed with ether. A few system plays a significant but complex role in the regulation of experiments were performed with eyes from cat, dog, monkey TOP. Sympathetic nerve fibers innervate the ciliary processes (Macaca fascicularl, obtained through the courtesy of the New and trabecular meshwork (1, 2), and both sympathetic stimu- England Primate Center), and human (obtained through the lation and locally applied fl-adrenergic agonists such as epi- courtesy of the New England Eye Bank). nephrine decrease lOP (2, 3). Paradoxically, /3-adrenergic an- Each eye was opened through a circumferential incision 3 tagonists such as timolol (which is effective clinically in treating mm posterior to the limbus, and the vitreous was bluntly dis- glaucoma) also decrease lOP (4). It has been suggested that sected from the posterior surface of the lens. The opened an- differences in sites of action (/3-adrenergic agonists facilitating terior third of the eye was placed, cornea down, in artificial reabsorptive mechanisms and f3-adrenergic antagonists de- aqueous humor (130 mM NaCI/2.7 mM KCI/18.3 mM creasing secretion) might explain this complex action of ad- NaH2CO3/1.33 mM MgCl2/1.5 mM CaC12/10 mM glucose/10 renergic agents on lOP (4, 5). However, biochemical studies, mM Hepes, pH 7.4), and the lens was removed by dividing the such as those measuring adrenergic receptor binding (6), so far zonules at the level of the lens capsule. The villus-like ciliary have not clearly indicated whether there are /3-adrenergic re- processes were freed from their attachments to the iris, tran- ceptors in the ciliary processes or trabecular meshwork separate sected at their attachments to the ciliary body, removed, washed from those in the neighboring iris, which also receives adren- in 150 mM NaCl, and homogenized (10 mg/ml) in 6 mM Tris ergic innervation. Furthermore, in the ciliary processes them- maleate buffer (pH 7.4). The iris was removed from its at- selves, it is not certain whether there are /-receptors in the se- tachment to the ciliary body, cleaned of any adhering ciliary cretory epithelium distinct from those on ciliary blood vessels. process tissue, washed, and homogenized as above. The ciliary Knowledge of such localization would be of value because it muscle was cleaned of any remaining ciliary process tissue, might help to understand whether the effect of adrenergic freed from the underlying sclera by blunt and sharp dissection antagonists in decreasing TOP is through an action on ciliary (care being taken not to remove any tissue from the area of the process blood flow or through a direct effect on secretion. trabecular meshwork), and then washed and homogenized as In general, further knowledge of the localization and char- above. Samples of all of the above tissues were taken prior to acteristics of 3-adrenergic receptors in secretory and reab- homogenization, fixed in 10% buffered formalin, and processed sorptive areas of the eye would be of potential benefit in the for routine light microscopy in order to determine the accuracy design of pharmacological agents for control of TOP. The of the dissections. present study attempted to determine: (i) if there are f- Separation of Ciliary Process Epithelial and Vascular Cells. The procedure used was adapted from previous methods The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "ad- Abbreviation: IOP; intraocular pressure. vertisement" in accordance with 18 U. S. C. §1734 solely to indicate * A preliminary report of some of these experiments has appeared this fact. [Nathanson, J. A. (1980) Soc. Neurosci. Abstr. 6,3561. 7420 Medical Sciences: Nathanson Proc. Natl. Acad. Sci. USA 77 (1980) 7421

(10, 11) used to isolate epithelial cells from the choroid plexus human (mean of two experiments). Histological examination (a tissue histologically similar to the ciliary processes). Specif- showed that the ciliary process of all species contained pigment ically, 90 mg of intact ciliary process obtained as described and secretory epithelium, vascular endothelium, some vascular above was placed in a small (3 ml) conical glass centrifuge tube smooth muscle and connective tissue cells, and intravascular in 2 ml of dissociation medium [10 mM Hepes, pH 7.4/137 mM blood elements. In some experiments in which rabbits were NaCl/2.7 mM KCI/0.7 mM Na2HCO3/5.6 mM glucose/0.1% perfused transcardially with phosphate-buffered saline to re- filtered trypsin (1:250) from GIBCO]. (A separate 15-mg sample move blood, stimulation of ciliary process adenylate cyclase of intact tissue was saved on ice for later comparison.) The tube activity by isoproterenol was similar to that seen without per- was incubated for 15 min at 350C with intermittent tituration, fusion. and then the tissue was allowed to settle; the supernatant, en- Compared to the rabbit ciliary process, rabbit ciliary body riched in suspended epithelial cells, was transferred to a cooled and rabbit iris showed much less stimulation by isoproterenol (40C) test tube, and horse serum was added to a final concen- (Fig. 1). For the ciliary body, Vmax was 16 + 5% of the Vmax tration of 10%. Fresh dissociation medium was added to the seen in the ciliary process; for iris, it was 22 1 3% of that for original tube containing sedimented tissue and the dissociation ciliary process (mean + SEM for three experiments, for each procedure was repeated twice more. The combined cell sus- tissue). The Ka for isoproterenol stimulation in iris and ciliary pensions from the three dissociations were filtered through a body varied between 1.4 and 4 X 10-7 M. Histologically, iris 100-,Mm-mesh nylon screen, sedimented at 100 X g for 5 min, and ciliary body contained mostly muscle tissue with a small and washed twice in the above medium lacking trypsin. One amount (estimated, <5%) of ciliary epithelium. In some ex- portion of the washed cell suspension was fixed in formalin for periments in which a greater amount of ciliary process epi- histologic study; another was used for determination of total thelium was present in the ciliary body dissections, there was cell count and proportion of cells excluding trypan blue, by a correspondingly greater stimulation of adenylate cyclase using standard hemocytometer techniques. The remainder of activity by isoproterenol, further supporting an enrichment of the cells were pelleted and homogenized (10 mg/ml) in 6 mM 3-adrenergic-sensitive adenylate cyclase in the ciliary pro- Tris maleate (pH 7.4) for determination of adenylate cyclase cess. activity. The remaining partially deepithelialized ciliary process Pharmacological Characteristics of jB-Adrenergic Stimu- vasculature and stroma was washed twice, a sample was taken lation in Ciliary Process. In the rabbit ciliary process, epi- for histologic study, and then the rest was homogenized (10 nephrine (Ka = 2.35 X 10-6 M) was at least 12 times more po- mg/ml) as above. tent than norepinephrine (Ka 22.9 X 10-5 M) in activating Adenylate Cyclase. Cyclic AMP formation was measured, adenylate cyclase (Fig. 2A). In three separate enzyme prepa- as described (10, 11), in 0.3 ml of 80 mM Tris maleate, pH rations, the average Vmax for these two compounds (at con- 7.4/10 mM theophylline/0.5 mM ethylene glycol bis(O-ami- centrations up to 1 mM) was about 30% less than the Vmax for noethyl ether)-N,N,N',N'-tetraacetic acid/8 mM MgCl2/0.03 isoproterenol. Both dopamine and the a-adrenergic agonist mM GTP/2 mM ATP (from equine muscle, with a trace of phenylephrine were much less effective than isoproterenol in vanadium) containing tissue homogenate (t0.7 mg wet weight) stimulating enzyme activity. This order of amine potency and test substances as indicated. Protein concentration was (isoproterenol > epinephrine > norepinephrine > phenyl- determined by the Lowry method with bovine serum albumin ephrine) is similar to that seen in vascular and bronchial smooth as a standard. Under the assay conditions used, enzyme activity muscle (32-enriched) compared to that found in heart, fat, and was linear with time up to at least 6 min and linear with respect small intestine (fll-enriched) (14). to protein concentration from half to twice that used in the In other experiments in which various combinations of standard incubation. Under these same conditions, cyclic nu- cleotide phosphodiesterase activity (12) was almost completely inhibited. 0 Inhibition constants (Ki) for the various adrenergic blockers 100- were calculated from the equation,Ki =(IC5o)/(l + S/Ka), in which IC5o is the concentration of antagonist necessary to give .> 50% inhibition of isoproterenol-stimulated activity, S is the >w e 80[ concentration of isoproterenol present, and Ka is the concen- c) .4 tration of isoproterenol (0.36 ,uM) necessary for half-maximal activation 60- of rabbit ciliary process adenylate cyclase activity e e) (13). CQ Drugs were obtained from the following sources: phentol- > 0o amine, CIBA-Geigy; zinterol, Mead Johnson; atenolol, Stuart 4OF Pharmaceuticals; prenalterol and H 35/25, Hassle; IPS 339, Hassle and G. LeClerc; practolol, J. Douglas; fluphenazine, 20~~ Squibb. 20

RESULTS a is i II I 0' -8 -7 -6 -5 -4 -3 Tissue Distribution. (-)-Isoproterenol, a /3-adrenergic log [isoproterenol I agonist, caused substantial stimulation of ciliary process enzyme FIG. 1. Comparative activities of isoproterenol-stimulated ade- activity. Under the standard incubation conditions, the Ka for nylate cyclase in rabbit ciliary process (@),iris (*), and ciliary body isoproterenol stimulation (mean + SEM for four experiments) (o). For each tissue, absolute stimulation above basal activity is ex- was 3.6 + 0.4 X 10-7 M, and maximal stimulation was 331 + pressed as a percentage of the maximal stimulation (at 0.1 mM iso- 44% of basal In the of other species proterenol) observed in the ciliary process. Values shown represent activity. ciliary process the mean + SEM for three separate experiments. Average basal ac- tested, maximal stimulation (Vmax) by isoproterenol was 206% tivities were (in pmol/mg of protein per min) 21.3 for ciliary process; in the cat (one experiment); 780% in the dog (one experiment); 17.9 for iris; and 22.4 for ciliary body. Average Vmaxfor isoproterenol 264% in the monkey (one experiment); and 1020% in the in the ciliary process was 79.4 pmol/mg of protein per min. 7422 Medical Sciences: Nathanson Proc. Natl. Acad. Sci. USA 77 (1980)

cd ciliary process enzyme activity or to block activation by iso- E 100 A proterenol (Fig. 3). Among the agonists, zinterol (K., 4 X 10-8 0 M) and OPC 2009 (Ka, 3 X 10-7 M), known to have relatively 801. high j3 selectivity (16, 17), were the most potent compounds ; tested. On the other hand, the j3I selective agent prenalterol (18) was almost ineffective in ._ 60 totally activating the enzyme. Al- Q 1. though zinterol and OPC 2009 were more potent than isopro- 40 terenol, neither resulted in as great a maximal stimulation. E 40 I. N Similar partial agonistic effects of zinterol and certain other 132 CU selective agents have been reported for isoproterenol-stimulated CU 20Il physiological responses in other tissues as well (19). C. Among the f3-adrenergic antagonists (Fig. 3B), the relatively C I4 selective 12 blockers IPS 339 (Ki, 3.2 X 10-9 M) and H35/25 U _.4 (Ki, 4.3 X 10-7 M) (20,21) were the most potent; the relatively -8 -7 -6 -5 -4 -3 selective #I blockers atenolol (Ki, 1.1 X 10-5 M) and practolol (Ki, 2.1 X 10-5 M) (19) were the least potent. Cellular Localization of Enzyme Activity. Because the ciliary process is a nonhomogeneous tissue, localization of E fl-adrenergic-sensitive adenylate cyclase within this organ 4-0 might give some idea as to which cell type(s) participates in 0 adrenergic regulation of aqueous humor production. For ex- ample, a preferential localization in ciliary vascular tissue would suggest noradrenergic regulation of ciliary blood flow or vascular permeability, whereas a localization in the epithelium might suggest a direct effect on aqueous humor secretion. ._co Qla *4 x 70 A E '.4 60 0 bR 50 log [antagonist] .4 FIG. 2. Effects of various catecholamine agonists (A) and an- *4 40 c tagonists (B) on rabbit ciliary process adenylate cyclase activity. (A) co Activity for each agonist is expressed as a percentage of the maximal stimulation observed with isoproterenol. (B) Stimulation (above that 30 seen in the presence of antagonist alone) is expressed as a percentage N of the stimulation seen in the presence of 3 ,uM isoproterenol alone. 0)GI 20 Values shown here and in Fig. 3 represent the mean + mean deviation for replicate samples each assayed for cyclic AMP in triplicate. ISO, 0L) 10 isoproterenol; EPI, epinephrine; NE, norepinephrine; PE, phenyl- a) /,' PRE -l ephrine; DA, dopamine; PROP, propranolol; TIM, timolol; FLU, UW I4- fluphenazine; PHENT, phentolamine. CU 0 -I - -8 -7 -6 -5 -4 -3 agonists (in optimal concentrations) were tested together (data not shown), there was no additivity between isoproterenol and 120 B epinephrine, norepinephrine, dopamine, or phenylephrine. In fact, all four of the latter drugs decreased isoproterenol-stim- %404 ulated enzyme activity, thus acting as partial agonists. These 0 data, combined with those from antagonist studies (below), W 80 suggest that the observed stimulation by these five amines is the *4 1; 60 result of the activation of the same class of receptors. *0 U The stimulation of ciliary process adenylate cyclase activity cU 40 by isoproterenol was inhibited by low concentrations of the '00) 1-adrenergic antagonist propranolol (calculated Ki, 1.4 X 10-9 M) and by the clinically effective antiglaucoma 13-antagonist 0 20 timolol (K1, 2.5 X 10-9 M) (Fig. 2B). The dopamine antagonist W/ fluphenazine was a weak inhibitor of isoproterenol activation 01 (Kj = 1.9 X 10-5 M) compared to its known potency at dopa- -8 -7 -6 -5 -4 -3 mine receptors (typically, K1 = 8 X 10-9 M) (15). The a- log [antagonist] adrenergic antagonist phentolamine caused only a small inhi- FIG. 3. Effects of selective i3,- and (32-adrenergic agonists (A) and bition of isoproterenol stimulation, even at high concentra- antagonists (B) on rabbit ciliary process adenylate cyclase activity. tions. (A) Activity for each agonist is expressed as a percentage of the To investigate the characteristics of the ciliary process 13- maximal stimulation observed with isoproterenol. (B) Stimulation (above that seen in the presence of antagonist alone) is expressed as adrenergic receptor in more detail, several 13-agonists and an- a percentage of the stimulation seen in the presence of 3 ,uM isopro- tagonists with relative selectivity for either #,/- or 32-adrenergic terenol alone. ZIN, zinterol; OPC, OPC 2009; PRE, prenalterol; receptors in other tissues were tested for their ability to stimulate ATEN, atenolol; PRAC, practolol. Medical Sciences: Nathanson Proc. Natl. Acad. Sci. USA 77 (1980) 7423 In order to localize enzyme activity, a dispersed cell fraction in Fig. 4D. Both basal and isoproterenol-stimulated activities containing epithelial cells was separated from the underlying were greater in the epithelial cell fraction than either in the vascular network by using controlled enzymatic digestion with whole ciliary process or in the partially de-epithelialized vas- low-dose trypsin. Combined with differential centrifugation cular network. and sieve filtration, this procedure resulted in a relatively uniform preparation of cells (80-90% trypan blue excluding) DISCUSSION that had an appearance in the light microscope identical to that These studies demonstrate the presence of a specific f3-adren- of intact secretory and pigment epithelium. Epithelial cells ergic-sensitive adenylate cyclase in the mammalian ciliary appeared singly, in pairs, and in small groups (Fig. 4C). In process. Pharmacological characterization reveals a pattern of unperfused animals there were also a small number of blood agonist and antagonist sensitivities similar to that seen in tissues elements. containing a predominance of f32-adrenergic receptors. To Fig. 4A shows the appearance of the ciliary process prior to quantitate this similarity further, the activation constants for treatment, and Fig. 4B shows the appearance after enzymatic isoproterenol, epinephrine, norepinephrine, and zinterol in digestion with trypsin. Some, but not all, of the darker staining stimulating ciliary process adenylate cyclase activity were secretory epithelium has been removed. Fig. 4C shows a group compared with published constants for the same agonists in of isolated secretory cells typical of those present in the epi- activating adenylate cyclase in membrane fractions from lung thelial cell fraction. Isoproterenol-stimulated enzyme activities and heart (22). (These and other recent studies have shown that in homogenates made from tissues shown in A-C are compared the majority of 13 receptors in mammalian lung are of a 12 type,

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_0~ a) 16C) _ E ._ " E a_ 0'120 o 2 0 *, E 80 > 40~

O0 C I C I C I C D Intact Vessels Cells FIG. 4. Isoproterenol-stimulated adenylate cyclase activity in "epithelial cell" and "vascular" fractions derived from rabbit ciliary process. (A) Intact ciliary process with a double layer of darker staining pigment and secretory epithelium. (B) After incubation with trypsin. In some areas of the micrograph (at left), the epithelium has been largely removed, leaving vascular and connective tissue cells; in other areas (at right), epithelium remains intact. (C) Group of cells present in the isolated "epithelial cell" fraction. The bottom portion of this cell group shows both layers of epithelium; at the top, only a single layer is present. (D) Basal (C) and isoproterenol-stimulated (I) adenylate cyclase activities in homogenates made from the fractions shown in A (Intact), B (Vessels), and C (Cells). Isoproterenol was 0.1 mM. Bar in each micrograph represents 25 Am. 7424 Medical Sciences: Nathanson Proc. Natl. Acad. Sci. USA 77 (1980) and the majority in mammalian heart are fli.) The calculated Finally, and of some clinical interest, the localization and correlation coefficient indicated an excellent correlation, r = characterization of 132-adrenergic-sensitive adenylate cyclase 0.99 (P < 0.01), between the agonist characteristics of the ciliary in the ciliary process epithelium have certain implications for and lung enzymes and a poor correlation, r = 0.30, between the the design of drugs potentially effective in decreasing IOP in ciliary process and heart enzymes. Similarly, the calculated normal and glaucomatous eyes. If factors such as drug ab- inhibitory constants (Ki) for practolol, atenolol, H35/25, IPS sorption and catabolism are taken into account, one would 339, and propranolol in blocking isoproterenol-sensitive ade- predict that potent 12-adrenergic antagonists should be as ef- nylate cyclase in the ciliary process were significantly correlated fective as nonspecific 1 blockers in decreasing IOP and, at the with the Ki values for these same antagonists in inhibiting iso- same time, have fewer potential side effects on tissues con- proterenol stimulation of lung (r = 0.92, P < 0.05) but not heart taining #1I receptors, such as the heart. Preliminary in vivo (r = 0.12) adenylate cyclase. studies comparing the effects of the nonspecific 13 blocker ti- These comparisons suggest that the majority of adenylate molol and the 12 antagonist IPS 339 appear to support this cyclase-associated adrenergic receptors in rabbit ciliary process possibility (25). are of a 132 type. Because a number of tissues appear to have a mixture of (3I and 12 subtypes (19, 22), a small percentage of Note Added in Proof. Consistent with the above findings, a recent ciliary #I-adrenergic receptors cannot be ruled out. Further- report has shown that rabbit ciliary process will specifically bind a more, it should be borne in mind that the results obtained here 3-adrenergic ligand (26). were with broken cell preparations and that in ivo drug selectivity may be influenced by other factors such as neuronal I thank Edward J. Hunnicutt for technical assistance. This work was and extraneuronal uptake mechanisms, susceptibility to de- supported by a grant from the William F. Milton Fund. gradative enzymes, and membrane solubility. Although the subtype of 1 receptor present in a given tissue may vary among 1. Ehinger, B. (1964) Acta Univ. Lund Sect. 2 20,3-23. different species (23), preliminary studies with postmortem 2. Sears, M. L. (1975) in Handbook of Physiology, Endocrinology human eyes (unpublished data) indicate that human ciliary VI, eds. Astwood, E. & Greep, R. (Am. Physiol. Soc., Washington, process also contains an adenylate cyclase with ,82-like char- DC), pp. 553-590. acteristics. 3. Davson, H. & Matchett, P. A. (1951) J. Physiol. (London) 113, The present data show an enrichment of 13-adrener- 389-397. gic-sensitive adenylate cyclase activity in the epithelial fraction 4. Zimmerman, T. J. & Boger, W. P., III. (1979) Surv. Opthalmol. prepared from ciliary process. This finding is consistent with 23,347-362. 5. Neufeld, A. H. (1979) Surv. Opthalmol. 23,363-370. the possibility that the adrenergic nervous system may affect 6. Neufeld, A. H. & Page, E. D. (1977) Invest. Ophthalmol. Visual aqueous humor production through a direct effect on epithelial Sci. 16, 1118-1124. cell secretory mechanisms. It suggests, also, that the epithelium 7. Robison, G. A., Butcher, R. W. & Sutherland, E. W. (1971) Cyclic may be the site of action of clinically effective 13-adrenergic AMP (Academic, New York). antagonists that decrease IOP. However, it is important to note 8. Wolfe, B. B., Harden, T. K. & Molinoff, P. B. (1977) Annu. Rev. that, although the epithelial cell fraction appeared to be largely Pharmacol. Toxicol. 17,575-604. free from vascular and other cell types by light microscopy, one 9. Minneman, K. P., Hedberg, A. & Molinoff, P. B. (1979) J. cannot entirely rule out the presence of a small number of en- Pharmacol. Exp. Ther. 211,502-508. dothelial cells or of attached cell fragments (such as nerve ter- 10. Nathanson, J. A. (1979) Science 204, 843-845. minals). Furthermore, because the ciliary process "vascular 11. Nathanson, J. A. (1980) Mol. Pharmacol. 18, 199-209. fraction" contained a mixture of cell types (endothelium, 12. Filburn, C. R. & Karn, J. (1973) Anal. Biochem. 52,505-516. smooth muscle cells, residual undissociated epithelium, blood 13. Cheng, Y.-C. & Prussoff, W. H. (1973) Biochem. Pharmacol. 22, elements, and nerves), it is difficult to say with certainty which 3099-3188. cell(s) contained the 1-adrenergic-sensitive adenylate cyclase 14. Lands, A. M., Arnold, A., McAuliff, J. P., Luduena, F. D. & in one Brown, T. G. (1967) Nature (London) 214, 4597-4598. activity observed this fraction, and cannot rule out the 15. Clement-Cormier, Y. C., Kebabian, J. W., Petzold, G. L. & possibility that the ciliary vasculature might also serve as a site Greengard, P. (1974) Proc. Natl. Acad. Sci. USA 71, 1113- for the action of adrenergic agents. 1117. In the present experiments, dissections of the rabbit trabe- 16. Temple, D. L., Comer, W. T., Ferguson, H. C. & Allen, L. E. cular meshwork did not yield preparations adequate for bio- (1976) J. Med. Chem. 19, 626-633. chemical analysis. However, in preliminary experiments in the 17. Himori, N. & Taira, N. (1977) Br. J. Pharmacol. 61, 9-17. monkey, it has been possible to obtain a preparation containing 18. Carlsson, E., Dahlof, C.-G., Hedberg, A., Persson, H. & Tang- approximately 50% trabecular tissue (most of the remainder strand, B. (1977) N.S. Arch. Pharmacol. 300, 101-105. being ciliary muscle). In homogenates of this preparation, 0.1 19. Minneman, K. G., Hegstrand, L. R. & Molinoff, P. B. (1979) Mol. mM isoproterenol caused a 193 + 7% stimulation of basal Pharmacol. 16, 21-33. adenylate cyclase activity, compared.to a 264 + 2% stimulation 20. Imbs, J. L., Miesch, F., Schwartz, J., Velly, J., LeClerc, G., Mann, by isoproterenol in monkey ciliary process. This activation is A. & Wermuth, C. G. (1977) Br. J. Pharmacol. 60, 357-362. of interest because it is known that adrenergic agonists and 21. Levy, B. & Wilkenfield, B. E. (1969) Eur. J. Pharmacol. 5, AMP can affect humor outflow in the 227-234. cyclic aqueous primate 22. Minneman, K. C., Hegstrand, L. R. & Molinoff, P. B. (1979) Mol. eye (24). It should be emphasized, however, that additional Pharmacol. 16,34-46. tissues, such as the venous plexuses draining the reabsorptive 23. Hancock, A. A., DeLean, A. L. & Lefkowitz, R. J. (1979) Mol. areas may also play a significant role in outflow. It will be of Phdrmacol. 16, 1-9. interest, in future biochemical studies, to determine if 13 re- 24. Neufeld, A. H. (1978) Exp. Eye Res. 27,387-397. ceptors are present in these other areas as well as to characterize 25. Nathanson, J. A. Br. J. Pharmacol., in press. further the 13-adrenergic receptor that may be present in the 26. Bromberg, B. B., Gregory, D. S. & Sears, M. L. (1980) Invest. trabecular meshwork. Ophthalmol. Visual Sci. 19, 203-206.