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Beta-adrenergic receptors regulating vascular smooth muscle tone are only localized to the intraocular segment of the long posterior ciliary artery in bovine eye
SURVEY OF O P H T H A L M O L O G Y VOLUME 39. SUPPLEMENT 1 9 MAY 1995
Beta-Adrenergic Receptors Regulating Vascular Smooth Muscle Tone Are Only Localized to the Intraocular Segment of the Long Posterior Ciliary Artery in Bovine Eye NIELS C. BERG N Y B O R G , MD, 1 DMScI, A N D PER
J.
NIELSEN, MD 2
1Department of Pharmacology, Aarhus Univers&,, Aarhus C, a~d ~-E~eDepartment, Rigshospitalet, Copenhagen,, Denmmk Abstract. Beta-adrenergic drugs are important drugs in glaucoma treatment. T h e i r exact mechanism of action is not yet fully understood but a decreased perfusion pressnre in the ciliary body due to blockade ofvasodilatory beta-adrenoceptors is thought to participate in the reduction ofintraocular pressure. This study investigates the vasodilator action of beta adrenergic-agents in intra- and extraocular arteries fi'om bovine and h u m a n eyes. Ring segments of retinal, choroidal, intraocular segment of long posterior ciliary artery, and segments of extraocular posterior ciliary arteries fi-om bovine eyes and short posterior ciliary arteries from freshly enucleated h u m a n eyes were mounted on an isometric myograph and their reactivity to beta-adrenergic drugs were studied. Of all vessel types examined only the intraocular segment of long posterior ciliary artery precontracted with prostaglandin F2a r e s p o n d e d to 1-isoprenaline (1 n M - 1 I~M) with a graded concentrationd e p e n d e n t relaxation (32 -+ 7%, n = 10) and a pD,_, of 7.5 +- 0.2 concentrations of lisoprenaline greater than 1 I*M induced contractions which were antagonized by a combined blockade of alpha l- and alpha2-adrenoceptors. Salbutamol (selective beta2-agonist) induced relaxations of similar magnitude as that ofl-isoprenaline but the sensitivity of the intraocular segment of long posterior ciliary arteries to salbutamol was 312 times less than that to 1-isoprenaline, pD,_, 7.2 -+ 0.2 and 4.4 _+ 0.1 (n = 6), respectively, whereas dobutamine (selective betal-agonist) had no effect between 1 nM and 10 p.M. Betaxolol (selective betal-antagonist) and ICI 115811 (selective beta2-antagonist) competitively antagonized isoprenaline-induced relaxations giving pKlvvalues of 5.9 -+ 0.1 (n = 11) and 8.5 _+ 0.1 (n = 6), respectively. T h e slope of the Schild-plots were equal to unity. Human posterior ciliary arteries with spontaneous tone or tone induced by 10 ~M PGF2~ did not react to isoprenaline (1 n M - 10 I~M). T h e bovine intraocular segment of long posterior ciliary arteries contracted to 1-noradrenaline without alpha-adrenoceptor blockade and addition of propranolol or cocaine did not change the concentration-response curve. 1-Phenylephrine and B-HT 933 induced both potent and strong contractions indicating the presence of both a l p h a l - and alpha2-adrenoceptnrs in these arteries. Although beta-adrenoceptors have been identified in various parts of the ocular circulation by radio-ligand binding techniques our data show that beta-adrenoceptors linked to regulation of vascular tone are only found in intraocular branches of the intraocular segment of long posterior ciliary artery in bovine eyes. The beta-adrenoceptors a p p e a r to belong to a single population ~)f beta2-adrenoceptors. A l p h a l - and alpha2-adrenoceptors present on the intraocular segment of long posterior ciliary artery smooth muscle interfere with the action of isoprenaline at high concentrations. The human posterior ciliary artery seems to lack beta-adrenoceptors coupled to f~)rce regulation. Anti-glaucomatous beta-adrenoceptor blocking drugs seem therefore to exert their vascular effect on the beta2-adrenoceptnrs in the feeding arteries of the ciliary body. (Surv O p h t h a l m o l 39 [Suppl 1, May]: $66-$75, 1995)
Key words, adrenaline * betaxolol 9 beta-adrenoceptors * bovine ocular arteries * dobutamine 9 human posterior ciliary arteries 9 ICI 118,551 9 isoprenoline 9 noradrenaline 9 salbu{amol $66
BETA-ADRENOCEPTORS IN OCULAR VESSELS
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Introduction
Materials and Methods
Treatment of primary open angle glaucomatous patients with high intraocular pressure (IOP) is to a great extent based on the application of eyedrops containing heta-adrenoceptor antagonist drugs, both nonselective and beta-1 selective?~'m The exact mechanism by which the reduction in IOP is brought about by these agents is not fully understood.'-"-' The IOP lowering effect is mediated by a blockade of beta-2adrenoceptors on the ciliary body nonpigmented epithelium causing a reduced production of a q u e o u s h u n l o r s}A~176 but outflow may be impeded as this is stimulated by beta-2-adrenoceptor stimulation with adrenaline.'-'"-':' The betaadrenoceptor antagonists may also increase alpha-adrenergic-induced vasoconstriction in the intraocular segment of long posterior ciliary artery and its branches in the ciliary body, 4~causing the perfusion pressure in the ciliary body to drop and, hence, the production of anterior chamber fluid. However, a reduced ocular blood f l o w 7"1'''23 may be an unwanted effect if this interferes with the nourishment of vital intraocular structures. Beta-adrenoceptors of the beta-2 subtype have been detected in abundance in the ciliary body and processes and, to some extent also in the choroid, but few beta-adrenoceptors have been detected in other areas of the eye. ~l-J:~,l:,.lSApplication of beta-adrenoceptor antagonists either locally or systemically has been linked with decreased blood flow in the ciliary body and processes and in the choroid. 7'1:''-':~However, changes in blood flow may be secondary to reduction of' the metabolic activity in the ciliary body and decreased autoregulation in the choroidal vasculature. ]~ Functional beta-adrenoceptors have to our knowledge never been demonstrated in isolated segments of ocular arteries or veins. Techniques have been developed for in vitro pharmacodynamic studies of arteries with diameters down to about 100 D m , ~6'~7 and we have therefore in the present experiments attempted to detect betaadrenoceptors regulating active force generation in intraocular (retinal, choroid, intraocular segment of long posterior ciliary artery feeding the ciliary body) and extraocular (short perioptic posterior ciliary and ophthalmic-ciliary arteries from the bovine eye and short posterior ciliary arteries from freshly enucleated human eyes) in order to further elucidate the possible vascular effects of beta-adrenoceptor antagonists used in antiglaucoma therapy.
Bovine eyes were obtained at the local slaughter house and transported to the laboratory in ice-cold physiological salt solution (PSS). Ring segments ofextraocular arteries were taken fi-om 1) a proximal ophthalmo-ciliary and 2) a perioptic part of the short posterior ciliary arteries feeding the retinal and perioptic choroidal circulation in the bovine eye (note that the cow eye does not have a central retinal artery and vein in the optic nerve as in humans). Intraocu[ar arteries were isolated from 3) the retina, 4) the choroid, and 5) the intraocular part of the intraocular segment of long posterior ciliary artery before it penetrates the ciliary body. Human short posterior ciliary arteries were isolated fi-om eyes enucleated due to intraocular malignant melanoma, hemorrhagic glaucoma and trauma. The studies on human tissue complied with the Helsinki declaration. The arteries were all mounted on 40 tzm diameter stainless steel wires fixed to mounting devices connected to a micrometer and a three transducer, respectively, for isometric wall tension measurements. '-'s q'he arteries were equilibrated in oxygenated (5cA CO,, in O,_,)bicarbonate buffer at 37~ pH 7.4 for 30 min before they were stretched to their optimal lumen diameter for active tension development, :~:~which corresponds to an intraluminal diameter 90(/} of that where the passive transmural pressure equals 100 mmHg. Before experimentation, the arteries were contracted repetitively with K-PSS (125 mM K +) until reproducible responses were recorded. The arteries were then contracted either with PGF,_,~ or with PSS containing 30 mM of K + (choroidal small arteries and proximal posterior ciliary arteries). The action of 1-isoprenaline and other adrenergic agonistic drugs was then determined by cumulative addition of the drugs to the organ bath when the tension development of the vessels had stabilized either in the presence of a precontractile agent or in normal bicarbonate buffer. For all concentration response curves an EC:,0 concentration (moles/litre, M) was calculated by fitting the responses (R) to the equation; R/R ...... = [A]'/([A] n + EC:,0")where A is the concentration of agonist and EC:,o is the concentration required to reach half maximal response, n is the Hill coefficient indicating the steepness of the concentration response curve. The sensitivity of the vessels to any agonist is given in terms of pD,_, values, where pD,_, is -log(EC:,0, [M]. In the bovine intraocular segment ofhmg pos-
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Surv Ophthahnol 39 (Suppl 1) May 1995 Proximal Posterior Ciliary Artery
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Fig. 1. Traces showing the effect of 1-isoprenaline without alpha-adrenergic bh)ckade in bovine isolated ocular arteries contracted with 30 mM K + (K:~0)or 10 >M prostaglandin F,,~ (P(;F,_,~). Concentration ofl-isoprenaline is given as log([M]), w indicates wash-out in drug-free PSS. Vertical scales show active vessel wall forces in miliNewton (raN), horizontal scales show time in minutes. Effective lumen diameter of the arteries are indicated in pan at the lower right of each trace.
terior ciliary arteries the b e t a - a d r e n o c e p t o r s were characterized f u r t h e r by d e t e r m i n i n g the r e c e p t o r affinity of the selective b e t a - a d r e n o c e p tot antagonists betaxolol (beta-l) a n d ICI 118,551 (beta-2). T h e affinity (-log(KL~[M]) was calculated as described by MacKay? ~ where
-log(Kl,[M] = log([antagonist], (M)) + log(CR1), w h e r e CR is concentration ratio d e t e r m i n e d as EC:,0 for agonist in p r e s e n c e of antagonist divided by EC:,. in absence of antagonist. F r o m the individual e x p e r i m e n t s a Schild-plot :~ was constructed. Plots of log(CR-1) against-log(antag-
B E T A - A D R E N O C E P T O R S IN O C U L A R VESSELS
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log(Concentration, [M]) Fig. 2. Effect of alpha-adrenoceptor antagonists on 1isoprenaline concentration response characteristics in bovine isolated intraocular segment of long posterior ciliary arteries. Responses have been normalized regarding the maximal relaxation induced by l-isoprenaline during first control curve. Circles: control curve; squares: 0.3 p~M phentolamine; triangles: 0.3 ~M prazosin and 10 p,M rauwolscine. Points represent mean of 6-8 vessels and vertical bars show _+ SEM where it exceeds the size of the symbol.
onist, [M] were analyzed using linear regression analysis.
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Fig. 3. Reproducibility of I-isoprenaline-induced relaxation in 6 bovine intraocular segment of king posterior ciliary arteries contracted with 10>M prostaglandin F2~. Experiments were made in the presence of 0.3 p~M prazosin and 10 p~M rauwolscine. Responses are normalized regarding maximal relaxing response to 1isoprenaline in the first control curve. Points show mean and vertical bars indicate + SEM where this value exceeds the size of the symbol.
sition of the c o m p o u n d . T h e vessels were incubated with antagonists tin" 15 min before any agonists were added. STATISTICS
S O L U T I O N S A N D DRUGS
PSS had the following composition in mM: NaCI 129, NaHCO:~ 25, KCI 4.7, MgSO4 1.2, KH,_,P()4 1.2, EDTA 0.026 and glucose 5.5. K-PSS containing 125 mM K + was c o m p o s e d as PSS except that NaCI was e x c h a n g e d with KCI on an e q u i m o l a r basis. Different concentrations o f K + were achieved by mixing a p p r o p r i a t e volumes of PSS and K-PSS to reach required concentration ofK. + Drugs used were l-isoprenaline H C I , 1-adrenaline H C I , l-noradrenaline H C I , 1-propranolol H C I (Sigma, St. Louis, MO, USA), betaxolol H C I (Alcon, Fort Worth, TX, USA), and ICI 118,551 H C I (ICI, UK). All drugs were dissolved and diluted in doubly distilled water and a d d e d to the o r g a n c h a m b e r in volumes not m o r e than 0 . 3 ~ to reach final required concentration. Exp e r i m e n t s with ICI 118,551 were p e r f o r m e d in a d a r k e n e d r o o m to avoid light-induced d e c o m p o -
Results are given as m e a n -+ SEM ( n u m b e r of vessels). Differences between means are comp a r e d using Students t-test's for paired or unpaired data where a p p r o p r i a t e . For all tests a pvalue less than 0.05 was considered significant.
Results
o f all vessels e x a m i n e d only the intraocular segment of |ong posterior ciliary artery f r o m the bovine eye r e s p o n d e d to the nonselective betaa d r e n o c e p t o r agonist 1-isoprenaline (1 n M - 1 ~M) with a g r a d e d c o n c e n t r a t i o n - d e p e n d e n t relaxation (32 -+ 7%, n = 10) when the arteries were precontracted with 10 ~M prostaglandin F~ (Fig. 1). Blockade of the a l p h a - a d r e n o c e p t o r s caused no a p p e a r a n c e o f b e t a - a d r e n o c e p t o r mediated relaxation in the nonresponsive ocular vessels, and only the contractile action of" l-isoprenaline in the perioptic posterior ciliary arter-
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Fig. 4. 1-Isoprenaline (circles), 1-noradrenaline (squares), and l-adrenaline (triangles) concentrationresponse curves in bovine intraocular ciliary arteries contracted with 10 IxM prostaglandin F,,~. Experiments were made in the presence of 10. Contractions at 10 IxM l-adrenaline and 1-noradrenaline are due to overriding the alpha-adrenoceptor blocking capacity of phentolamine. Responses are calculated as change in wall tension just prior to addition ofagonist. Points represent mean of 10-12 vessels and vertical bars show -+ SEM where this value exceeds the size of the symbol.
Fig. 5. Dobutamine (betal-adrenoceptor selective agonist, filled squares), salbutamol (beta2-adrenoceptot selective agonist, filled triangles), and 1-isoprenaline (non-selective beta-1 and beta-2-adrenoceptor agonist, circles) concentration-response curves in bovine isolated intraocular anterior ciliary arteries. Responses have been normalized in relation to the maximal relaxation induced by isoprenaline. Points represent mean of 6 vessels and vertical bars show-+ SEM where this value exceeds the size of the symbol. Contraction to l-isoprenaline at 10 I.tM is due to absence of alpha-adrenergic blocking drugs.
ies was antagonized by 10 IxM phentolamine, a nonselective a l p h a - a d r e n o c e p t o r antagonist (results not shown). Papaverine (not shown) induced a full relaxation of all vessels tested. T h e pDe for 1-isoprenaline was 7.5 + 0.2 (10). Concentrations of 1-isoprenaline greater than 1 IxM induced contractions which were antagonized by a combined blockade of alpha-l- and alpha-2-adrenoceptors (Fig. 2), indicating that 1isoprenaline has secondary effects mediated t h r o u g h alpha-adrenoceptors. T h e relaxation of the intraocular segment of long posterior ciliary arteries induced by 1-isoprenaline was reproducible in four consecutive concentration-response experiments (Fig. 3), indicating that determination of antagonist binding affinity (Schild-plots) can be made without correction for t i m e - d e p e n d e n t drift in ECs0. T h e vascular b e t a - a d r e n o c e p t o r is not subjected to inactivation d u r i n g e x p o s u r e to 1-isoprenaline. 4 1-adrenaline and 1-noradrenaline (both alphaand b e t a - a d r e n o c e p t o r agonists) induced con-
c e n t r a t i o n - d e p e n d e n t relaxations of similar m a g n i t u d e as t-isoprenaline (Fig. 4) in vessels incubated with 10 IxM p h e n t o l a m i n e and contracted with 10 IxM prostaglandin F,_,~.T h e o r d e r of potency was adrenaline > isoprenaline -> noradrenaline. T h e naturally occurring catecholmines had a more p r o n o u n c e d contractile action than 1-isoprenaline in the highest concentration range due to their stronger alpha-adrenergic activity. Salbutamol (selective beta2-adrenergic agonist) induced relaxations similar in m a g n i t u d e to that o f 1-isoprenaline (117 +- 32%, n = 6), but the sensitivity o f the intraocular segment of long posterior ciliary arteries to salbutamol was 312 times less than that to 1-isoprenaline, pD,_, 7.2 + 0.2 and 4.4 -+ 0.1 (n = 6) p < 0.01, respectively. Dobutamine (selective betal-agonist) had no effect between 1 nM and 10 IxM (Fig. 5). Betaxolol (selective beta- 1-antagonist) and I C I 115.811 (selective beta-2-antagonist) antagonized 1-isoprenaline-induced relaxations corn-
BETA-ADRENOCEPTORS IN OCULAR VESSELS
Fig. 6. Schild-plots for ICI 118,511 (selective beta-2-adrenoceptor antagonist, closed circles) and betaxolol (selective beta-l-adrenoceptor antagonist, open circles) in bovine isolated intraocular segment of long posterior ciliary arteries using l-isoprenaline as agonist. Alpha-adrenoceptors were antagonized by 10 txM phantolamine. Points represent mean of 10-11 vessels and vertical bars show _+ SEM where this value exceeds the size of the symbol. Slopes of regression lines and their' intercepts with the x-axis (pA,_,-values) are indicated in figure.
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petitively giving pKu-values of 5.9 -+ 0.1 (n = 11) and 8.5 +-0.1 (n = 6), respectively. T h e slope of the Schild-plots were equal to unity (Fig. 6) for both antagonists over the concentration ranges tested. Without p r e i n d u c e d tone l-noradrenaline caused a c o n c e n t r a t i o n - d e p e n d e n t contraction of intraocular segment of long posterior ciliary arteries kept in PSS, pD..,-value 7.0 +- 0.1 (n = 6). 1-phenylephrine (selective alpha-l-adrenoceptor agonist) and B - H T 933 (selective aipha-2a d r e n o c e p t o r agonist) both induced concentrat i o n - d e p e n d e n t concentrations reaching similar maximal responses as seen with 1-noradrenaline; however, the selective alpha-adrenoceptor ago-
Fig. 7. l-Noradrenaline (alpha-1 and alpha-2-adrenoceptor agonist, open circles), l-phenylephrine (alpha ladrenoceptor selective agonist, filled triangles) and B-HT 933 (alpha-2-adrenoceptor selective agonist, filled squares) concentration-response curves in bovine isolated intraocular segment of long posterior ciliary arteries. Points represent mean of 6 vessels and vertical bars show _+ SEM where this value exceeds the size of the symboh
nists were less potent than 1-noradrenaline, pD,,values: 6.0 -+ 0.1 (n = 6 ) a n d 5.5 -+ 0.2 (n = 6), respectively (Fig. 7). T h e 1-noradrenaline concentration-response curve was not a u g m e n t e d by addition 3 IxM propranolol nor was the concentration-response curve shifted to the left (results not shown). Blockade of the neuronal uptake mechanism of amines with 3 IxM cocaine did not a u g m e n t the sensitivity of the intraocular segment of long posterior ciliary arteries to 1-noradrenaline (Fig. 8). Fig. 9 shows the trace of one isolated h u m a n posterior short ciliary artery exhibiting spontaneous tone exposed to 1-isoprenaline in a cumulative manner. Similar lack of dilating and con-
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Fig. 8. Effect of 3 txM cocaine (filled circles) on 1-noradrenaline concentration-response characteristics in bovine isolated intraocular segment of long posterior ciliary arteries. Second control curve after wash-out of cocaine is shown by squares. Points represent mean of 6 vessels and vertical bars show _+ SEM where this value exceeds the size of the symbol.
NYBORG, NIELSEN
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tractile action of l-isoprenaline was observed in six vessels from six different patients having spontaneous tone or being contracted with 10 IxM prostaglandin F~.~.
Discussion The present results show that beta-adrenoceptors linked to vascular smooth muscle force regulation only were detectable in isolated segments of the intraocular segment of long posterior ciliary artery in bovine eyes. None of the other intraocular and extraocular arteries from the bovine eye and the human isolated posterior short ciliary arteries we examined responded with relaxations to stimulation with |-isoprenaline; thus, the force-regulating vascular beta-adrenoceptors seem to be inhomogeneous distributed within the ocular circulation. A non-homogeneous receptor distribution causing regional heterogeneous reaction of vessels to noradrenaline, calcitonin gene-related peptide and atrial natriuretic factor is often encountered. ~':~e3~We have previously shown that angiotensin II only has a contractile action in bovine ciliary arteries and not in intraocular retinal small arteries? 4 The regional differences in receptor distribution within the ocular circulation is not fully investigated, but in other organs as well one may find dramatic changes in localization of receptors such as in the coronary circulation in rats where the endothelial alpha-2adrenoceptor :~ and CGRP-receptor disappears approximately 0.5 cm distal to the origin of the left coronary arteryY Age has also been shown to influence the ex-
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pression ofbeta-adrenergic receptors in vascular tissue. It is well known that the aortic beta-2adrenoceptors disappear within the first two months and two years of life in rats and rabbits, respectively. '4 We have used eyes from cows older than one year and therefore we cannot exclude that the ocular arteries in which we find a void of functional beta-adrenoceptors actually had a beta-adrenoceptor population linked to force regulation when the cows were younger. Beta-adrenoceptors of the beta-2 subtype have previously been demonstrated in the eye using radioligand binding and autoradiographic methods."-l:~"~'17 The highest number of binding sites and strongest staining is found particularly in and over the ciliary body and related tiss u e J 2"17 Beta-adrenoceptor binding sites have been demonstrated in bovine retinal arteries ':~ and rabbit choroidal tissue, '~ but we and others have not been able to link these receptors to the force regulating processes in bovine retinal '6':~' and choroidal arteries (present study) having diameters between 150-300 Ixm. This apparent lack of effect ofbeta-adrenoceptor agonists could be due to a more distal location of the betaadrenoceptors in arteries too small for isolation with the current techniques. The discrepancy between binding and present functional studies shows the necessity of coupling several techniques including perfusion studies for the full understanding of pharmacological characteristic of any particular vascular bed or tissue. The ocular beta-adrenoceptor subtype has been characterized in binding and autoradiographic studies and all studies indicate the pres-
BETA-ADRENOCEPTORS IN OCULAR VESSELS
ence of beta-2-adrenoceptor subtype ~'v-''~:''17 except in retinal arteries where an equal distribution of beta-1 and beta-2-adrenoceptors are found. ~:~However, the ocular beta-2-adrenoceptor appears to exhibit less stereoselectivity between d- and 1-forms of ligands than other peripheral beta-adrenoceptors. '-'~ Our study indicates that the beta-adrenoceptors on the bovine intraocular segment of long posterior ciliary artery all belong to the beta-2-adrenoceptor subtype since the slopes of the Schild-plots were equal to unity :~''-'t and the affinities for the selective beta-1-and beta-2-adrenoceptor antagonists were comparable to values obtained for these agents in other tissues with recognized populations of the beta-2-adrenoceptor subtype. TM The order of potency of the selective beta-adrenoceptot agonists, dobutamine and salbutamol, also indicate the presence of beta-2-adrenoceptors since only salbutamol induced a relaxation. The potency of salbutamol was, however, very low compared to that ofl-isoprenaline. The order of potency of adrenaline, noradrenaline and isoprenaline was different than expected, when looked upon in relation to the data obtained with the selective antagonists, as we would expect isoprenaline to be more potent (higher pD2) than adrenaline and noradrenaline. Adrenaline was, however, more potent than noradrenaline, thus fulfilling the classical criteria for the beta-2-adrenoceptor. Thus the beta-adrenoceptor on the bovine intraocular segment of long posterior ciliary artery is not typical but most of its pharmacology indicates that it is a beta-2-adrenoceptor and our results are thus in agreement with the previous indirect binding studies. The human posterior ciliary arteries we examined did not respond to I-isoprenaline, indicating that the possibly present beta-adrenoceptors are too few in number to influence force generation. Our observation is in part supported by previous experiments on postmortem human posterior ciliary arteries, showing a small nonsignificant relaxation and a contraction at the highest concentration of isoprenaline used. 45 The small effect of beta-adrenoceptor stimulation in the human posterior ciliary artery could be explained by a rapid desensitization of the betaadrenoceptors during the cumulative addition of isoprenaline. 4This explanation must be tested in future experiments. The intraocular segment of long posterior ciliary artery also had a population ofalpha-adrenergic receptors mediating a strong contraction. The alpha-adrenoceptors seem to dominate
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functionally over the beta-adrenoceptors, at least in vitro, because l-noradrenaline only induced contractions in these arteries with (not shown) and without tone, and propranolol did not augment the maximal contraction nor the sensitivity of the vessels to 1-noradrenaline. In the rabbit, however, application of timolol or betaxolol caused localized constriction a2 of arterioles in the ciliary processes without affecting the downstream vessel caliber, indicating that beta-adrenoceptors may be dominating in certain regions in the ciliary vasculature. This again shows that direct studies on isolated vessel segments may give little information about pharmacology of vessels further downstream. Both phenylephrine (alphal-adrenoceptor agonist) and B-HT 933 (alpha2-adrenoceptor agonist) induced potent contractions in the intraocular segment of long posterior ciliary arteries, strongly indicating that both alpha-1 and alpha-2 adrenoceptors are present in these vessels. It has previously been difficult to demonstrate alpha-2-adrenoceptors in isolated vessel preparations, although perfusion studies clearly showed their existence in the circulation. It is now realized that the alpha-2-adrenoceptors are found in the smaller arteries and their importance for contraction is inversely related to the vessel caliber, at least in humans? ~There may be exceptions to this rule in the eye, as we and others ~6:~ have shown that bovine retinal arteries only have contractile alpha-l-adrenoceptors al-
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Surv Ophthalmol 39 (Suppl 1) May 1995
though the diameter is only half that of the intraocular segment of long posterior ciliary arteries. The l-noradrenaline-induced contraction was not changed by cocaine. This suggests that the sympathetic innervation in these arteries is sparse or that the distance between the nerves and vascular smooth muscle cells is wide. Previous experiments on the dog ophthahnic artery and posterior ciliary arteries have shown a marginal effect of uptake-1 inhibitors like imipramine and cocaine. :~r':"~ From a clinical point of view, our results indicate that beta-adrenoceptor blockade may be associated with a decreased ciliary blood flow. 7"~:''4'-' The intraocular concentration of both selective beta-1 and nonselective beta-adrenoceptor antagonists used in antiglaucoma treatment has been shown to be sufliciently high to cause a complete blockade of the ciliary beta-2- adrenoceptors:~7':~s'43; thus, the difference in efficacy and longterm clinical outcome of the selective and nonselective beta-adrenoceptor antagonists used in antiglaucoma treatment must depend on effects other than their beta-adrenoceptor blocking and IOP lowering effect per se. Although the IOP in patients with glaucoma can be clinically controlled, it may not necessarily indicate a normal ocular blood flow. The ability of the betaadrenoceptor antagonists used in antiglaucoma treatment not to interfere with the ocular blood flow therefore becomes important. Some betaadrenoceptor antagonists exhibit intrinsic activity and others also have direct vasodilating effects due to a blockade of calcium channels in the vascular smooth muscle cells. 6 It may be speculated that additional therapeutic effects of some betaadrenoceptor antagonists s''-'4 may be brought about by a direct vasodilatory action on the ocular circulation increasing the blood flow to important ocular structures, such as the optic nerve head. In conclusion, we have shown that beta-adrenoceptors of the beta-2 subtype are located only in segments of the intraocular segment of long posterior ciliary artery, the main feeding artery of the ciliary body vasculature, in bovine eyes. Inhibition of the vascular beta-2-adrenoceptors wilt likely reduce the blood flow and perfusion pressure in the ciliary body and this effect could be additive to the effect of the beta-adrenoceptor antagonists on the aqueous humor production in the ciliary epithelium. The present data can not determine the relative weight of each action of beta-adrenergic antiglaucomatous drugs.
NYBORG, NIELSEN
References 1. Aalkjaer C, Mulvany MJ, Nyborg NCB: Atrial natriuretic tactor causes specific relaxation of l'al renal al'Cll~tle arteries. B~ .] Pharmacol 86:447-453, 1985 2. Alvarado JA, Franse Ca,man L, McHolm (,, Murphy C: Epinephrine eit~ects on tm'~jor cell types of the aqueous outflow pathway: in vitro studies/clinical implications. 7"rmc~ Am Ophlhalmol Soc 88: 267-282, 1990 3. Arunlakshana O, Schild HO: Some quantitative uses of drug antagonists. B~,] Pharmacol 14:48-58, 1{159 4. BenovicJL, Stone WC, Caron MG, Letkowitz RJ: Inhibition of the beta-adrenergic receptor kinase by polyanions. J Biol Chem 264:67117-6710, 1989 5. Bilski A I, Halliday SE, FitzgeraldJ D, WaleJ L: The pharmacology of a beta 2-selective adrenoeeptor antagonist (ICI 118,551 )..] Cardiovasc Pharmacol 5:430-437, 1983 6. Boonen HC, Struyker Boudiev HA, De Me}.lt,: EtR'cts of tertatoH on the responsiveness of isolated femoral, lnesenteric, and renal resistance arteries to adrenergic stimuli..] Cardiovasc F'ha~macol 15:124-129, 1990 7. Chiou (;C, Chen Y.I: Eli;eels of antiglaucoma drugs on o(:ular blood flow in ocular hypertensive rabbits../O(ul Pharmacol 9.'13-24, 1993 8. Collignon Brach.l: Long-term eftcct of ophthalmic betaadrenoceptor antagonists o n illtraoc/tlal" pressure alld retinal sensitivity in primary open-angle glaucoma. (.'urr Eve Res 11:1-3, 1992 9. Danyluk AW, Paton D: Diagnosis and management of glaucoma. Clm Syrup 43:2-32, 1991 10. Dutlsworth TS: Drug treatnlellt o['glau(;ollla. ,Ira Plutrm NS33:43-52, 1993 1 I. Elena PP, Denis P, Kosina Boix M, et al: Beta adrenergic binding sites in tile human eye: an autoradiographic study..] Ocul Pharmacol 6:143-149, 1990 12. Elena PP, Kosina Boix M, Moulin (;, Lapalus P: Autol'adiographic localization of beta-adrenergic receptors in rabbit eye. t,ve.sl ()pbfbahmd l "is Sci 28:1436-144 I, 1987 13. Ferra,'i I)ileo (;: Beta 1 and beta 2 adrenergic binding sites in bovine retina and retinal blood vessels, ht-,,e,,I Ophlhahmd Vi~ Sci 29:695-699, 1988 14. Fleich.IH, Maling M H, Brodie BB: Beta-receptor activity on aorta. Circ Res 26:151-162, 1970 15. G'rajewski AL, Ferrari Dileo G, Feuer Wj, Anderson DR: Beta-adrenergic responsiveness of choroidal vasculature. Ophlhahnolog 7 98:989-995, 1{191 16. Hoste AM, Boels PJ, Andries LJ, ct al: Elti_,cts of beta.antagonists on contraction of bovine retinal micvoarteries in vitro, h*-ees! Ophlhahmd I'is Sci 31:1231-1237, 1990 17. Jampel HI), Lynch M(;, Brown RH, et al: Beta-adrenmgic receptors in human tvabecular meshwork, ldentilicalion and autoradiographic Io(alizalion. l,vesl Ophlhahmd l'is Sci 28:772-779, 1987 18. Kahle (,, Kaulen P, Wollensak.l: Quantitative autoradiogt-aphy ofbeta-adre,mrgic receptors in l'abl)it eyes. E,~./) Eve Re~ 51.'503-507, 1990 19. l~iel .I W, Shepherd AP: Autoregulation of choroidal blood tlow in the rabbit, hr~,e.~l ()phlh(thmd l'i~ Sci 33: 2399-2410, 1992 20. Koike K, Hagiwara H, "lakayanagi I: Comparison o['interactions of R-( + )- and S-( - )-isolners of beta-adrenergic partial agonists, I)eiim()H and carteoH, with high altinity site of I)eta-adrcn(wcptors in isolated rabl)it ciliary body and guinea-pig taenia cacti+ Cm~.] Pbv.~iol F'tm~macol 6 c):9.")1-{)57, 1991 21. Mackay D: H(m should ,,alues otpA2 and a[tinit)((instants ti)Y pharlnacological competitive al|Iagonists I)C estimated? .] Pharm Phm macol 70:312-313, 1978 22. Martin X: Mechanism ()f action otbeta-bl(wkers. Ophlalmolog'ie 4."154-155, 1990 23. Martin XI), Rabineal~ PA: Vasoconstrictive ella:el o[ topical timoh)l on hulnan retinal arteries. (h'aCfi,~ A*rh Clm
BETA-ADRENOCEPTORS IN OCULAR VESSELS Exp Ophlhalmol 227:526-5"~0, 1989 24. Messmer C, FlannnerJ, Stumpfig D: Influence ufbetaxulol and timolol on the visual fields of patients with glaucoma. Am J Ophlhahnol 112:678-681, 1991 25. Mishima S: Ocular effects ofbeta-adrenergic agents. XII Jules Stein Lecture. Surv Ophthalmol 27.'187-208, 1982 26. Mulvany MJ, Aalkjzer C: Structure and function of small arteries. Am J Physiol 70.'921-961, 1990 27. Mulvany MJ, Halpern W: Mechanical properties of vascular smooth muscle in situ. Nature 260.'617-619, 1976 28. Mulvany MJ, Nyborg NCB: An increased calcium sensitivity of mesenteric resistance vessels in spontaneously hypertensive rats. BrJ Pharmacol 71.'585-596, 1980 29. Nathanson JA: 8tereospecificity of beta adrenergic antagunists: R-enantiomers show increased selectivity for hera-2 receptors in ciliary process.J Pha~vnacolExp Ther 245:94-101, 1988 30. Nielsen H, Mortensen FV, Mulvany MJ: Differential distributinn of postjunctional alpha 2 adrenoceptors in human omental small arteries. J Cardiovasc Pharmacol 16." 34-40, 1990 31. Nielsen PJ, Nyborg NCB: Adrenergic responses in isolated bovine retinal resistance arteries, lnt Ophthalmol 13.'103-107, 1989 32. Nyborg NCB: Action ofnoradrenaline on isolated proximal and distal coronary arteries of rat: selective release of enduthelium-derived relaxing factor in proximal arteries. Br ] Pharmacol 100.'552-556, 1990 33. Nyborg NCB, Korsgaard N, Nielsen PJ: Active wall tension-length curve and morphology of isolated bovine retinal small arteries: important feature for pharmacodynalnic studies. Exp E';e Res 51:217-224, 1990 34. Nyburg NCB, Nielsen PJ, Prieto D, Benedito S: Angiotensin II does nut contract bovine retinal resistance arteries in vitro. Exp Eye Res 50:469-474, 1990 35. Ohkubu H, Chiha S: Regional differences of vascular sensitivities in canine long posterior ciliary arteries. Curr L'x,e Res 7.'457-463, 1988 36. Ohkubo H, Chiba S: Vascular responses of ophthalmic arteries to exogenous and endogenous norepinephrine. Exp Eve Res 48.'539-547, 1989 37. Phan TM, Nguyen KP, GiacominiJC, Lee DA: Ophthalmic beta-bh~ckers: determination of plasma and aqueous humor levels 6"r a radioreceptor assay following multiple
$75 doses. J Ocul Pharmacol 7.'243-252, 1991 38. PulanskyJ, Friedman Z, Fauss D, et ah Effects ofbetaxolol/timolol on epinephrine stimulated cyclic-AMP levels in human trabecular meshwork cells, lnt Ophthalmol 13.-95-97, 1989 39. Prieto D, Benedito S, Nyburg NCB: Heterogeneous involvement of endothelium in calcitonin gene-related peptide-induced relaxation in coronary arteries from rat. BrJ Pharmacol 103.'1764-1768, 1991 40. Reiss GR, Brubaker RF: The mechanism ofbetaxolol, a new ocular hypotensive agent. Ophthalmology 90.'13691372, 1983 41. Rimele TJ, Henry DE, Giesa FR, et al: Comparison of the beta-adreuoceptor affinity and selectivity of cetamolol, atenolol, betaxolol, and ICI-118,551. J Cardiovasc Pharmacol 12:208-217, 1988 42. Van Buskirk EM, Bacon DR, Fahrenbach WH: Ciliary vasoconstriction after topical adrenergic drugs. Am J Ophlhahnol 109.'511-517, 1990 43. Vuuri ML, Ali Melkkila T, Kaila T, et ah Plasma and aqueous humour concentrations and systemic effects of topical betaxolol and timolol in man. Acta Ophthalmol Copenh 71.'201-206, 1993 44. Yabhmski ME, Novack GD, Burke PJ, et al: The effect of levobunulol on aqueous humor dynamics. Exp Eye Res 44.'49-54, 1987 45. Yu DY, Alder VA, Su EN, et al: Agunist response of human isolated posterior ciliary artery. Invest Ophthalmol ['is Sci 33.'48-54, 1992
Supported in part by Landsforeningen til Bek~empelse af Ojensygdumme og Blindhed, Danish Health Research Council (,rant No 12-8717 and Aarhus University. This work is part of the EURAD (European Working Party on Resistance Artery Diseases) Project uf the Commission of the European Union and Health Research Programme. None of the authors or members of their families have any proprietary interests in any product used in this study. Reprint address: Dr. Niels C. Berg Nyborg, Department of Pharmacology, Aarhus, University, DK-8000 Aarhus C, Denmark.
Beta-Adrenergic Receptors Regulating Vascular Smooth Muscle Tone Are Only Localized to the Intraocular Segment of the Long Posterior Ciliary Artery in Bovine Eye NIELS C. BERG N Y B O R G , MD, 1 DMScI, A N D PER
J.
NIELSEN, MD 2
1Department of Pharmacology, Aarhus Univers&,, Aarhus C, a~d ~-E~eDepartment, Rigshospitalet, Copenhagen,, Denmmk Abstract. Beta-adrenergic drugs are important drugs in glaucoma treatment. T h e i r exact mechanism of action is not yet fully understood but a decreased perfusion pressnre in the ciliary body due to blockade ofvasodilatory beta-adrenoceptors is thought to participate in the reduction ofintraocular pressure. This study investigates the vasodilator action of beta adrenergic-agents in intra- and extraocular arteries fi'om bovine and h u m a n eyes. Ring segments of retinal, choroidal, intraocular segment of long posterior ciliary artery, and segments of extraocular posterior ciliary arteries fi-om bovine eyes and short posterior ciliary arteries from freshly enucleated h u m a n eyes were mounted on an isometric myograph and their reactivity to beta-adrenergic drugs were studied. Of all vessel types examined only the intraocular segment of long posterior ciliary artery precontracted with prostaglandin F2a r e s p o n d e d to 1-isoprenaline (1 n M - 1 I~M) with a graded concentrationd e p e n d e n t relaxation (32 -+ 7%, n = 10) and a pD,_, of 7.5 +- 0.2 concentrations of lisoprenaline greater than 1 I*M induced contractions which were antagonized by a combined blockade of alpha l- and alpha2-adrenoceptors. Salbutamol (selective beta2-agonist) induced relaxations of similar magnitude as that ofl-isoprenaline but the sensitivity of the intraocular segment of long posterior ciliary arteries to salbutamol was 312 times less than that to 1-isoprenaline, pD,_, 7.2 -+ 0.2 and 4.4 _+ 0.1 (n = 6), respectively, whereas dobutamine (selective betal-agonist) had no effect between 1 nM and 10 p.M. Betaxolol (selective betal-antagonist) and ICI 115811 (selective beta2-antagonist) competitively antagonized isoprenaline-induced relaxations giving pKlvvalues of 5.9 -+ 0.1 (n = 11) and 8.5 _+ 0.1 (n = 6), respectively. T h e slope of the Schild-plots were equal to unity. Human posterior ciliary arteries with spontaneous tone or tone induced by 10 ~M PGF2~ did not react to isoprenaline (1 n M - 10 I~M). T h e bovine intraocular segment of long posterior ciliary arteries contracted to 1-noradrenaline without alpha-adrenoceptor blockade and addition of propranolol or cocaine did not change the concentration-response curve. 1-Phenylephrine and B-HT 933 induced both potent and strong contractions indicating the presence of both a l p h a l - and alpha2-adrenoceptnrs in these arteries. Although beta-adrenoceptors have been identified in various parts of the ocular circulation by radio-ligand binding techniques our data show that beta-adrenoceptors linked to regulation of vascular tone are only found in intraocular branches of the intraocular segment of long posterior ciliary artery in bovine eyes. The beta-adrenoceptors a p p e a r to belong to a single population ~)f beta2-adrenoceptors. A l p h a l - and alpha2-adrenoceptors present on the intraocular segment of long posterior ciliary artery smooth muscle interfere with the action of isoprenaline at high concentrations. The human posterior ciliary artery seems to lack beta-adrenoceptors coupled to f~)rce regulation. Anti-glaucomatous beta-adrenoceptor blocking drugs seem therefore to exert their vascular effect on the beta2-adrenoceptnrs in the feeding arteries of the ciliary body. (Surv O p h t h a l m o l 39 [Suppl 1, May]: $66-$75, 1995)
Key words, adrenaline * betaxolol 9 beta-adrenoceptors * bovine ocular arteries * dobutamine 9 human posterior ciliary arteries 9 ICI 118,551 9 isoprenoline 9 noradrenaline 9 salbu{amol $66
BETA-ADRENOCEPTORS IN OCULAR VESSELS
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Introduction
Materials and Methods
Treatment of primary open angle glaucomatous patients with high intraocular pressure (IOP) is to a great extent based on the application of eyedrops containing heta-adrenoceptor antagonist drugs, both nonselective and beta-1 selective?~'m The exact mechanism by which the reduction in IOP is brought about by these agents is not fully understood.'-"-' The IOP lowering effect is mediated by a blockade of beta-2adrenoceptors on the ciliary body nonpigmented epithelium causing a reduced production of a q u e o u s h u n l o r s}A~176 but outflow may be impeded as this is stimulated by beta-2-adrenoceptor stimulation with adrenaline.'-'"-':' The betaadrenoceptor antagonists may also increase alpha-adrenergic-induced vasoconstriction in the intraocular segment of long posterior ciliary artery and its branches in the ciliary body, 4~causing the perfusion pressure in the ciliary body to drop and, hence, the production of anterior chamber fluid. However, a reduced ocular blood f l o w 7"1'''23 may be an unwanted effect if this interferes with the nourishment of vital intraocular structures. Beta-adrenoceptors of the beta-2 subtype have been detected in abundance in the ciliary body and processes and, to some extent also in the choroid, but few beta-adrenoceptors have been detected in other areas of the eye. ~l-J:~,l:,.lSApplication of beta-adrenoceptor antagonists either locally or systemically has been linked with decreased blood flow in the ciliary body and processes and in the choroid. 7'1:''-':~However, changes in blood flow may be secondary to reduction of' the metabolic activity in the ciliary body and decreased autoregulation in the choroidal vasculature. ]~ Functional beta-adrenoceptors have to our knowledge never been demonstrated in isolated segments of ocular arteries or veins. Techniques have been developed for in vitro pharmacodynamic studies of arteries with diameters down to about 100 D m , ~6'~7 and we have therefore in the present experiments attempted to detect betaadrenoceptors regulating active force generation in intraocular (retinal, choroid, intraocular segment of long posterior ciliary artery feeding the ciliary body) and extraocular (short perioptic posterior ciliary and ophthalmic-ciliary arteries from the bovine eye and short posterior ciliary arteries from freshly enucleated human eyes) in order to further elucidate the possible vascular effects of beta-adrenoceptor antagonists used in antiglaucoma therapy.
Bovine eyes were obtained at the local slaughter house and transported to the laboratory in ice-cold physiological salt solution (PSS). Ring segments ofextraocular arteries were taken fi-om 1) a proximal ophthalmo-ciliary and 2) a perioptic part of the short posterior ciliary arteries feeding the retinal and perioptic choroidal circulation in the bovine eye (note that the cow eye does not have a central retinal artery and vein in the optic nerve as in humans). Intraocu[ar arteries were isolated from 3) the retina, 4) the choroid, and 5) the intraocular part of the intraocular segment of long posterior ciliary artery before it penetrates the ciliary body. Human short posterior ciliary arteries were isolated fi-om eyes enucleated due to intraocular malignant melanoma, hemorrhagic glaucoma and trauma. The studies on human tissue complied with the Helsinki declaration. The arteries were all mounted on 40 tzm diameter stainless steel wires fixed to mounting devices connected to a micrometer and a three transducer, respectively, for isometric wall tension measurements. '-'s q'he arteries were equilibrated in oxygenated (5cA CO,, in O,_,)bicarbonate buffer at 37~ pH 7.4 for 30 min before they were stretched to their optimal lumen diameter for active tension development, :~:~which corresponds to an intraluminal diameter 90(/} of that where the passive transmural pressure equals 100 mmHg. Before experimentation, the arteries were contracted repetitively with K-PSS (125 mM K +) until reproducible responses were recorded. The arteries were then contracted either with PGF,_,~ or with PSS containing 30 mM of K + (choroidal small arteries and proximal posterior ciliary arteries). The action of 1-isoprenaline and other adrenergic agonistic drugs was then determined by cumulative addition of the drugs to the organ bath when the tension development of the vessels had stabilized either in the presence of a precontractile agent or in normal bicarbonate buffer. For all concentration response curves an EC:,0 concentration (moles/litre, M) was calculated by fitting the responses (R) to the equation; R/R ...... = [A]'/([A] n + EC:,0")where A is the concentration of agonist and EC:,o is the concentration required to reach half maximal response, n is the Hill coefficient indicating the steepness of the concentration response curve. The sensitivity of the vessels to any agonist is given in terms of pD,_, values, where pD,_, is -log(EC:,0, [M]. In the bovine intraocular segment ofhmg pos-
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Fig. 1. Traces showing the effect of 1-isoprenaline without alpha-adrenergic bh)ckade in bovine isolated ocular arteries contracted with 30 mM K + (K:~0)or 10 >M prostaglandin F,,~ (P(;F,_,~). Concentration ofl-isoprenaline is given as log([M]), w indicates wash-out in drug-free PSS. Vertical scales show active vessel wall forces in miliNewton (raN), horizontal scales show time in minutes. Effective lumen diameter of the arteries are indicated in pan at the lower right of each trace.
terior ciliary arteries the b e t a - a d r e n o c e p t o r s were characterized f u r t h e r by d e t e r m i n i n g the r e c e p t o r affinity of the selective b e t a - a d r e n o c e p tot antagonists betaxolol (beta-l) a n d ICI 118,551 (beta-2). T h e affinity (-log(KL~[M]) was calculated as described by MacKay? ~ where
-log(Kl,[M] = log([antagonist], (M)) + log(CR1), w h e r e CR is concentration ratio d e t e r m i n e d as EC:,0 for agonist in p r e s e n c e of antagonist divided by EC:,. in absence of antagonist. F r o m the individual e x p e r i m e n t s a Schild-plot :~ was constructed. Plots of log(CR-1) against-log(antag-
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sition of the c o m p o u n d . T h e vessels were incubated with antagonists tin" 15 min before any agonists were added. STATISTICS
S O L U T I O N S A N D DRUGS
PSS had the following composition in mM: NaCI 129, NaHCO:~ 25, KCI 4.7, MgSO4 1.2, KH,_,P()4 1.2, EDTA 0.026 and glucose 5.5. K-PSS containing 125 mM K + was c o m p o s e d as PSS except that NaCI was e x c h a n g e d with KCI on an e q u i m o l a r basis. Different concentrations o f K + were achieved by mixing a p p r o p r i a t e volumes of PSS and K-PSS to reach required concentration ofK. + Drugs used were l-isoprenaline H C I , 1-adrenaline H C I , l-noradrenaline H C I , 1-propranolol H C I (Sigma, St. Louis, MO, USA), betaxolol H C I (Alcon, Fort Worth, TX, USA), and ICI 118,551 H C I (ICI, UK). All drugs were dissolved and diluted in doubly distilled water and a d d e d to the o r g a n c h a m b e r in volumes not m o r e than 0 . 3 ~ to reach final required concentration. Exp e r i m e n t s with ICI 118,551 were p e r f o r m e d in a d a r k e n e d r o o m to avoid light-induced d e c o m p o -
Results are given as m e a n -+ SEM ( n u m b e r of vessels). Differences between means are comp a r e d using Students t-test's for paired or unpaired data where a p p r o p r i a t e . For all tests a pvalue less than 0.05 was considered significant.
Results
o f all vessels e x a m i n e d only the intraocular segment of |ong posterior ciliary artery f r o m the bovine eye r e s p o n d e d to the nonselective betaa d r e n o c e p t o r agonist 1-isoprenaline (1 n M - 1 ~M) with a g r a d e d c o n c e n t r a t i o n - d e p e n d e n t relaxation (32 -+ 7%, n = 10) when the arteries were precontracted with 10 ~M prostaglandin F~ (Fig. 1). Blockade of the a l p h a - a d r e n o c e p t o r s caused no a p p e a r a n c e o f b e t a - a d r e n o c e p t o r mediated relaxation in the nonresponsive ocular vessels, and only the contractile action of" l-isoprenaline in the perioptic posterior ciliary arter-
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Fig. 4. 1-Isoprenaline (circles), 1-noradrenaline (squares), and l-adrenaline (triangles) concentrationresponse curves in bovine intraocular ciliary arteries contracted with 10 IxM prostaglandin F,,~. Experiments were made in the presence of 10. Contractions at 10 IxM l-adrenaline and 1-noradrenaline are due to overriding the alpha-adrenoceptor blocking capacity of phentolamine. Responses are calculated as change in wall tension just prior to addition ofagonist. Points represent mean of 10-12 vessels and vertical bars show -+ SEM where this value exceeds the size of the symbol.
Fig. 5. Dobutamine (betal-adrenoceptor selective agonist, filled squares), salbutamol (beta2-adrenoceptot selective agonist, filled triangles), and 1-isoprenaline (non-selective beta-1 and beta-2-adrenoceptor agonist, circles) concentration-response curves in bovine isolated intraocular anterior ciliary arteries. Responses have been normalized in relation to the maximal relaxation induced by isoprenaline. Points represent mean of 6 vessels and vertical bars show-+ SEM where this value exceeds the size of the symbol. Contraction to l-isoprenaline at 10 I.tM is due to absence of alpha-adrenergic blocking drugs.
ies was antagonized by 10 IxM phentolamine, a nonselective a l p h a - a d r e n o c e p t o r antagonist (results not shown). Papaverine (not shown) induced a full relaxation of all vessels tested. T h e pDe for 1-isoprenaline was 7.5 + 0.2 (10). Concentrations of 1-isoprenaline greater than 1 IxM induced contractions which were antagonized by a combined blockade of alpha-l- and alpha-2-adrenoceptors (Fig. 2), indicating that 1isoprenaline has secondary effects mediated t h r o u g h alpha-adrenoceptors. T h e relaxation of the intraocular segment of long posterior ciliary arteries induced by 1-isoprenaline was reproducible in four consecutive concentration-response experiments (Fig. 3), indicating that determination of antagonist binding affinity (Schild-plots) can be made without correction for t i m e - d e p e n d e n t drift in ECs0. T h e vascular b e t a - a d r e n o c e p t o r is not subjected to inactivation d u r i n g e x p o s u r e to 1-isoprenaline. 4 1-adrenaline and 1-noradrenaline (both alphaand b e t a - a d r e n o c e p t o r agonists) induced con-
c e n t r a t i o n - d e p e n d e n t relaxations of similar m a g n i t u d e as t-isoprenaline (Fig. 4) in vessels incubated with 10 IxM p h e n t o l a m i n e and contracted with 10 IxM prostaglandin F,_,~.T h e o r d e r of potency was adrenaline > isoprenaline -> noradrenaline. T h e naturally occurring catecholmines had a more p r o n o u n c e d contractile action than 1-isoprenaline in the highest concentration range due to their stronger alpha-adrenergic activity. Salbutamol (selective beta2-adrenergic agonist) induced relaxations similar in m a g n i t u d e to that o f 1-isoprenaline (117 +- 32%, n = 6), but the sensitivity o f the intraocular segment of long posterior ciliary arteries to salbutamol was 312 times less than that to 1-isoprenaline, pD,_, 7.2 + 0.2 and 4.4 -+ 0.1 (n = 6) p < 0.01, respectively. Dobutamine (selective betal-agonist) had no effect between 1 nM and 10 IxM (Fig. 5). Betaxolol (selective beta- 1-antagonist) and I C I 115.811 (selective beta-2-antagonist) antagonized 1-isoprenaline-induced relaxations corn-
BETA-ADRENOCEPTORS IN OCULAR VESSELS
Fig. 6. Schild-plots for ICI 118,511 (selective beta-2-adrenoceptor antagonist, closed circles) and betaxolol (selective beta-l-adrenoceptor antagonist, open circles) in bovine isolated intraocular segment of long posterior ciliary arteries using l-isoprenaline as agonist. Alpha-adrenoceptors were antagonized by 10 txM phantolamine. Points represent mean of 10-11 vessels and vertical bars show _+ SEM where this value exceeds the size of the symbol. Slopes of regression lines and their' intercepts with the x-axis (pA,_,-values) are indicated in figure.
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-5
-4
log(Antagonist, [M])
petitively giving pKu-values of 5.9 -+ 0.1 (n = 11) and 8.5 +-0.1 (n = 6), respectively. T h e slope of the Schild-plots were equal to unity (Fig. 6) for both antagonists over the concentration ranges tested. Without p r e i n d u c e d tone l-noradrenaline caused a c o n c e n t r a t i o n - d e p e n d e n t contraction of intraocular segment of long posterior ciliary arteries kept in PSS, pD..,-value 7.0 +- 0.1 (n = 6). 1-phenylephrine (selective alpha-l-adrenoceptor agonist) and B - H T 933 (selective aipha-2a d r e n o c e p t o r agonist) both induced concentrat i o n - d e p e n d e n t concentrations reaching similar maximal responses as seen with 1-noradrenaline; however, the selective alpha-adrenoceptor ago-
Fig. 7. l-Noradrenaline (alpha-1 and alpha-2-adrenoceptor agonist, open circles), l-phenylephrine (alpha ladrenoceptor selective agonist, filled triangles) and B-HT 933 (alpha-2-adrenoceptor selective agonist, filled squares) concentration-response curves in bovine isolated intraocular segment of long posterior ciliary arteries. Points represent mean of 6 vessels and vertical bars show _+ SEM where this value exceeds the size of the symboh
nists were less potent than 1-noradrenaline, pD,,values: 6.0 -+ 0.1 (n = 6 ) a n d 5.5 -+ 0.2 (n = 6), respectively (Fig. 7). T h e 1-noradrenaline concentration-response curve was not a u g m e n t e d by addition 3 IxM propranolol nor was the concentration-response curve shifted to the left (results not shown). Blockade of the neuronal uptake mechanism of amines with 3 IxM cocaine did not a u g m e n t the sensitivity of the intraocular segment of long posterior ciliary arteries to 1-noradrenaline (Fig. 8). Fig. 9 shows the trace of one isolated h u m a n posterior short ciliary artery exhibiting spontaneous tone exposed to 1-isoprenaline in a cumulative manner. Similar lack of dilating and con-
Z ,(wD
I
i
-9
-8
i
i
-7 -6 log(Concentration,
i
-5 [M]}
i
!
-4
-3
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Surv Ophthalmol 39 (Suppl 1) May 1995
Fig. 8. Effect of 3 txM cocaine (filled circles) on 1-noradrenaline concentration-response characteristics in bovine isolated intraocular segment of long posterior ciliary arteries. Second control curve after wash-out of cocaine is shown by squares. Points represent mean of 6 vessels and vertical bars show _+ SEM where this value exceeds the size of the symbol.
NYBORG, NIELSEN
I
-9
tractile action of l-isoprenaline was observed in six vessels from six different patients having spontaneous tone or being contracted with 10 IxM prostaglandin F~.~.
Discussion The present results show that beta-adrenoceptors linked to vascular smooth muscle force regulation only were detectable in isolated segments of the intraocular segment of long posterior ciliary artery in bovine eyes. None of the other intraocular and extraocular arteries from the bovine eye and the human isolated posterior short ciliary arteries we examined responded with relaxations to stimulation with |-isoprenaline; thus, the force-regulating vascular beta-adrenoceptors seem to be inhomogeneous distributed within the ocular circulation. A non-homogeneous receptor distribution causing regional heterogeneous reaction of vessels to noradrenaline, calcitonin gene-related peptide and atrial natriuretic factor is often encountered. ~':~e3~We have previously shown that angiotensin II only has a contractile action in bovine ciliary arteries and not in intraocular retinal small arteries? 4 The regional differences in receptor distribution within the ocular circulation is not fully investigated, but in other organs as well one may find dramatic changes in localization of receptors such as in the coronary circulation in rats where the endothelial alpha-2adrenoceptor :~ and CGRP-receptor disappears approximately 0.5 cm distal to the origin of the left coronary arteryY Age has also been shown to influence the ex-
-8
-7 log (Noradrenaline,
!
I
-6
-5
[M] )
pression ofbeta-adrenergic receptors in vascular tissue. It is well known that the aortic beta-2adrenoceptors disappear within the first two months and two years of life in rats and rabbits, respectively. '4 We have used eyes from cows older than one year and therefore we cannot exclude that the ocular arteries in which we find a void of functional beta-adrenoceptors actually had a beta-adrenoceptor population linked to force regulation when the cows were younger. Beta-adrenoceptors of the beta-2 subtype have previously been demonstrated in the eye using radioligand binding and autoradiographic methods."-l:~"~'17 The highest number of binding sites and strongest staining is found particularly in and over the ciliary body and related tiss u e J 2"17 Beta-adrenoceptor binding sites have been demonstrated in bovine retinal arteries ':~ and rabbit choroidal tissue, '~ but we and others have not been able to link these receptors to the force regulating processes in bovine retinal '6':~' and choroidal arteries (present study) having diameters between 150-300 Ixm. This apparent lack of effect ofbeta-adrenoceptor agonists could be due to a more distal location of the betaadrenoceptors in arteries too small for isolation with the current techniques. The discrepancy between binding and present functional studies shows the necessity of coupling several techniques including perfusion studies for the full understanding of pharmacological characteristic of any particular vascular bed or tissue. The ocular beta-adrenoceptor subtype has been characterized in binding and autoradiographic studies and all studies indicate the pres-
BETA-ADRENOCEPTORS IN OCULAR VESSELS
ence of beta-2-adrenoceptor subtype ~'v-''~:''17 except in retinal arteries where an equal distribution of beta-1 and beta-2-adrenoceptors are found. ~:~However, the ocular beta-2-adrenoceptor appears to exhibit less stereoselectivity between d- and 1-forms of ligands than other peripheral beta-adrenoceptors. '-'~ Our study indicates that the beta-adrenoceptors on the bovine intraocular segment of long posterior ciliary artery all belong to the beta-2-adrenoceptor subtype since the slopes of the Schild-plots were equal to unity :~''-'t and the affinities for the selective beta-1-and beta-2-adrenoceptor antagonists were comparable to values obtained for these agents in other tissues with recognized populations of the beta-2-adrenoceptor subtype. TM The order of potency of the selective beta-adrenoceptot agonists, dobutamine and salbutamol, also indicate the presence of beta-2-adrenoceptors since only salbutamol induced a relaxation. The potency of salbutamol was, however, very low compared to that ofl-isoprenaline. The order of potency of adrenaline, noradrenaline and isoprenaline was different than expected, when looked upon in relation to the data obtained with the selective antagonists, as we would expect isoprenaline to be more potent (higher pD2) than adrenaline and noradrenaline. Adrenaline was, however, more potent than noradrenaline, thus fulfilling the classical criteria for the beta-2-adrenoceptor. Thus the beta-adrenoceptor on the bovine intraocular segment of long posterior ciliary artery is not typical but most of its pharmacology indicates that it is a beta-2-adrenoceptor and our results are thus in agreement with the previous indirect binding studies. The human posterior ciliary arteries we examined did not respond to I-isoprenaline, indicating that the possibly present beta-adrenoceptors are too few in number to influence force generation. Our observation is in part supported by previous experiments on postmortem human posterior ciliary arteries, showing a small nonsignificant relaxation and a contraction at the highest concentration of isoprenaline used. 45 The small effect of beta-adrenoceptor stimulation in the human posterior ciliary artery could be explained by a rapid desensitization of the betaadrenoceptors during the cumulative addition of isoprenaline. 4This explanation must be tested in future experiments. The intraocular segment of long posterior ciliary artery also had a population ofalpha-adrenergic receptors mediating a strong contraction. The alpha-adrenoceptors seem to dominate
$73
Isoprenaline -9 9
-5 @
9
9
9
s~,o.~ mM 9
5 mN
5 rain Fig. 9. Trace showing effect ofl-isoprenaline (1 nM- 10 p.M) on human isolated 314 p,m diameter short posterior ciliary artery exhibiting spontaneous tone isolated from 76-yr-old female. Concentration of isoprenaline in given as log([M]). SNP is sodium nitroprusside. Vertical scale shows vessel wall force in milliNewton. Horizontal scale shows time in rain.
functionally over the beta-adrenoceptors, at least in vitro, because l-noradrenaline only induced contractions in these arteries with (not shown) and without tone, and propranolol did not augment the maximal contraction nor the sensitivity of the vessels to 1-noradrenaline. In the rabbit, however, application of timolol or betaxolol caused localized constriction a2 of arterioles in the ciliary processes without affecting the downstream vessel caliber, indicating that beta-adrenoceptors may be dominating in certain regions in the ciliary vasculature. This again shows that direct studies on isolated vessel segments may give little information about pharmacology of vessels further downstream. Both phenylephrine (alphal-adrenoceptor agonist) and B-HT 933 (alpha2-adrenoceptor agonist) induced potent contractions in the intraocular segment of long posterior ciliary arteries, strongly indicating that both alpha-1 and alpha-2 adrenoceptors are present in these vessels. It has previously been difficult to demonstrate alpha-2-adrenoceptors in isolated vessel preparations, although perfusion studies clearly showed their existence in the circulation. It is now realized that the alpha-2-adrenoceptors are found in the smaller arteries and their importance for contraction is inversely related to the vessel caliber, at least in humans? ~There may be exceptions to this rule in the eye, as we and others ~6:~ have shown that bovine retinal arteries only have contractile alpha-l-adrenoceptors al-
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Surv Ophthalmol 39 (Suppl 1) May 1995
though the diameter is only half that of the intraocular segment of long posterior ciliary arteries. The l-noradrenaline-induced contraction was not changed by cocaine. This suggests that the sympathetic innervation in these arteries is sparse or that the distance between the nerves and vascular smooth muscle cells is wide. Previous experiments on the dog ophthahnic artery and posterior ciliary arteries have shown a marginal effect of uptake-1 inhibitors like imipramine and cocaine. :~r':"~ From a clinical point of view, our results indicate that beta-adrenoceptor blockade may be associated with a decreased ciliary blood flow. 7"~:''4'-' The intraocular concentration of both selective beta-1 and nonselective beta-adrenoceptor antagonists used in antiglaucoma treatment has been shown to be sufliciently high to cause a complete blockade of the ciliary beta-2- adrenoceptors:~7':~s'43; thus, the difference in efficacy and longterm clinical outcome of the selective and nonselective beta-adrenoceptor antagonists used in antiglaucoma treatment must depend on effects other than their beta-adrenoceptor blocking and IOP lowering effect per se. Although the IOP in patients with glaucoma can be clinically controlled, it may not necessarily indicate a normal ocular blood flow. The ability of the betaadrenoceptor antagonists used in antiglaucoma treatment not to interfere with the ocular blood flow therefore becomes important. Some betaadrenoceptor antagonists exhibit intrinsic activity and others also have direct vasodilating effects due to a blockade of calcium channels in the vascular smooth muscle cells. 6 It may be speculated that additional therapeutic effects of some betaadrenoceptor antagonists s''-'4 may be brought about by a direct vasodilatory action on the ocular circulation increasing the blood flow to important ocular structures, such as the optic nerve head. In conclusion, we have shown that beta-adrenoceptors of the beta-2 subtype are located only in segments of the intraocular segment of long posterior ciliary artery, the main feeding artery of the ciliary body vasculature, in bovine eyes. Inhibition of the vascular beta-2-adrenoceptors wilt likely reduce the blood flow and perfusion pressure in the ciliary body and this effect could be additive to the effect of the beta-adrenoceptor antagonists on the aqueous humor production in the ciliary epithelium. The present data can not determine the relative weight of each action of beta-adrenergic antiglaucomatous drugs.
NYBORG, NIELSEN
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$75 doses. J Ocul Pharmacol 7.'243-252, 1991 38. PulanskyJ, Friedman Z, Fauss D, et ah Effects ofbetaxolol/timolol on epinephrine stimulated cyclic-AMP levels in human trabecular meshwork cells, lnt Ophthalmol 13.-95-97, 1989 39. Prieto D, Benedito S, Nyburg NCB: Heterogeneous involvement of endothelium in calcitonin gene-related peptide-induced relaxation in coronary arteries from rat. BrJ Pharmacol 103.'1764-1768, 1991 40. Reiss GR, Brubaker RF: The mechanism ofbetaxolol, a new ocular hypotensive agent. Ophthalmology 90.'13691372, 1983 41. Rimele TJ, Henry DE, Giesa FR, et al: Comparison of the beta-adreuoceptor affinity and selectivity of cetamolol, atenolol, betaxolol, and ICI-118,551. J Cardiovasc Pharmacol 12:208-217, 1988 42. Van Buskirk EM, Bacon DR, Fahrenbach WH: Ciliary vasoconstriction after topical adrenergic drugs. Am J Ophlhahnol 109.'511-517, 1990 43. Vuuri ML, Ali Melkkila T, Kaila T, et ah Plasma and aqueous humour concentrations and systemic effects of topical betaxolol and timolol in man. Acta Ophthalmol Copenh 71.'201-206, 1993 44. Yabhmski ME, Novack GD, Burke PJ, et al: The effect of levobunulol on aqueous humor dynamics. Exp Eye Res 44.'49-54, 1987 45. Yu DY, Alder VA, Su EN, et al: Agunist response of human isolated posterior ciliary artery. Invest Ophthalmol ['is Sci 33.'48-54, 1992
Supported in part by Landsforeningen til Bek~empelse af Ojensygdumme og Blindhed, Danish Health Research Council (,rant No 12-8717 and Aarhus University. This work is part of the EURAD (European Working Party on Resistance Artery Diseases) Project uf the Commission of the European Union and Health Research Programme. None of the authors or members of their families have any proprietary interests in any product used in this study. Reprint address: Dr. Niels C. Berg Nyborg, Department of Pharmacology, Aarhus, University, DK-8000 Aarhus C, Denmark.