Exp. Eye Res. (1990)

50. 469-474

Angiotensin NIELS

C. BERG

II Does NYBORG”,

Not Contract Bovine Arteries In Vitro

PER J. NIELSEN”,

DOLORES

Retinal PRIET06

AND

Resistance SARA BENEDlTO*

Department of Pharmacology, Aarhus University DK-8000 Aarhus C, “Eye Department, Hjwring Sygehus, DK-9800 Hjwring, Denmark, and bDepartment of Physiology, Veterinary Faculty, Universidad Complutense de Madrid, 28040 Madrid, Spain (Received 29 May 1989 and accepted in revised form 23 October 1989) II was studied in vitro on ring segments of bovine retinal resistancearteries(i.d. 126-271 ,um) and posterior ciliary arteries (i.d. 207-1153 pm). Although the retinal resistance arterieswere responsiveto 5-hydroxytryptamine, prostaglandinF,,, and changesin extracellular K’concentration,they did not, in contrast to the posteriorciliary arteries,contract to cumulative or single dosesof angiotensinII. In the latter arteries,angiotensinII induceda smallconcentration dependent contraction, 5% of maximal 125 mM K+-inducedresponse,with a pD,-value of 9.3. The singleaddition of lo-’ M angiotensinII increasedthe maximalvesselresponseof the posteriorciliary arteriesthree times to angiotensinII. Tachyphyiaxis waspronouncedin the posteriorciliary arteries,in which the response to angiotensinII could not be repeated.Indomethacin( 10e5M). methyleneblue (3 x 1O-6M), or removal of endotheliumdid not makethe retinal resistancearteriesresponsiveto angiotensinII. Retinal arteries precontractedwith 30 mMpotassiumdid not respondto angiotensinII. AngiotensinII did not potentiate the 5-hydroxytryptamine- and noradrenaline concentration-response characteristicsof both retinal resistanceand posteriorciliary arteries.Although angiotensinII-receptorshave beendetectedin bovine retinal vascular smoothmuscleusing radioligand-bindingtechnique.the presentresultssuggestthat The effect of angiotensin

these receptors are non-functional in respect to regulation of retinal resistance artery tone. Key words: retinal resistancearteries: posteriorciliary arteries; angiotensinII: 5-hydroxytryptamine :

noradrenaline; bovine.

1. Iutroduction Angiotensin II (octapeptide), being formed from angiotensin I (decapeptide) by angiotensin converting enzyme (ACE), is an important regulator of several physiological functions, among those regulation of blood pressure and intra-vascular volume (Re, 1984). It has been suggested that locally produced or blood-borne vasoactive substanceslike angiotensin II may interfere with the autoregulatory capacity of the retinal and optic disc vasculature, making the eye more or less vulnerable to the development of glaucomatous visual Eeld defects (Sossiand Anderson, 1982). High amounts of ACE have been found in both choroidal and retinal vessels(Ward et al. I979 ; Igic and Kojovic, 1980; Ferrari-Dileo et al.. 1988) and angiotensin II receptors detected by specific radioligand binding technique (Ferrari-Dileo, Davis and Anderson, 1987) are also present in human as well as bovine retinal vessels,thus suggesting that the retinal vascular tone may be regulated by angiotensin II. In accordance with this assumption, in&a-arterial injection of angiotensin II in human volunteers (Dollery, Hill and Hodge, 1963) as well as i.v. injection in cats have been reported to constrict retinal vessels(Rockwood et al., 1987). * For correspondence and reprint requests. 0014+?35/90/050469+06

$03.00.0

A study by Alm (1972), in contrast, did not show change in the oxygen tension close to the retina after intra-arterial injection of angiotensin II in cats. It is thus still unclear whether angiotensin II does affect retinal vascular tone or not. This question can be answered, at least in part, by direct studies on the action of angiotensin II on isolated retinal vascular segments. We have recently shown that bovine retinal arteries with an internal diameter of > 130 pm can be isolated and studied as ring segmentsin vitro on an isometric myograph (Nielsen and Nyborg, 1989a,b). The present experiments were initiated to test if the angiotensin IIreceptors present on the bovine retinal and posterior ciliary arteries (Ferrari-Dileo et al., 1987) are functionally active regarding regulation of vascular smooth muscle tone. 2. Materials and Methods Retinal resistance arteries and posterior ciliary arteries were isolated from enucleated bovine eyes (variable domestic strains, b.w. 350-500 kg), as previously described (Nielsen and Nyborg, 1989a,b) and mounted as rings on an isometric myograph, which allowed direct measurement of wall force while the internal circumference could be controlled (Mulvany and Halpern, 1976 ; Mulvany and Nyborg, 1980). The vesselswere kept, unless otherwise stated, 0 1990 AcademicPressLimited

470

N. C. B NYBORG

ETAL

response induced by concentration, A. of angiotensin II relative to maximal vessel response induced by angiotensin II, EC,,, is concentration of angiotensin I1 required to give half maximal response, and 17is the ’ Hill-coefficient’ or slope factor. Extra concentration response experiments were made with retinal resistance arteries being preincubated with lo-” M indomethacin (Sigma, St Louis, MO), 3 x 10efi M methylene blue (DAK, Copenhagen. Denmark) and finally, with vessels devoid of endothelium. This was removed by inserting a scalp hair through the lumen of the vessel and then gently pushing the hair back and forth about eight times and rotating the vessel on the mounting wires to secure removal of endothelium along the whole internal surface of the vessel. Removal of endothelium was tested by the absence of acetylcholine-induced relaxation of vessels precontracted with 10 ~3;\r prostaglandin F,, (Dinoprost’R, Upjohn, S.A. Puurs. Belgium). Retinal and posterior ciliary arteries were in a separate series of experiments, challenged only with one high concentration, 1O-6 M. of angiotensin II to avoid tachyphylaxis during construction of normal concentration response curves. The putative potentiating effect of angiotensin 11 was further tested on the 5-hydroxytryptamine ( 5-HT HCl. serotonin, Sigma) and 1-noradrenaline [ 1 -noradrenaline ( 1-arterenol) HCl, Sigma] concentration effect curves. Vessel responses are given as active tension (Newton

in oxygenated (5% CO, in 0,) physiological saline solution (PSS) with the following composition in mM: NaCI, 119: NaH,CO,, 25: KCI, 4.7: CaCl,. 1.5: KH,PO,, 1.18: MgSO,, 1.17; EDTA, 0026: glucose, 11 (analytical grade from E. Merck, A.G.. Darmstadt, F.R.G.). The vessels were equilibrated at 37 “C, pH 7.4, for 30 min, and were then stretched to their optimal lumen diameter, I,, for active tension development, corresponding to 90% of the diameter the vessels would have had when subjected to a transmural pressure of 13.3 kPa (100 mmHg) and relaxed (for further detail see Nyborg et al., 1988: Nielsen and Nyborg, 1989a). The effective normalized lumen diameter was in the range of 126-2 71 pm for the retinal resistance arteries, and 207-l 15 3 /rm for the posterior ciliary arteries. The arteries were subsequently activated repetitively with a high potassium containing solution (12 5 mM) K-PSS (similar to PSS except that NaCl was exchanged for KC1 on an equimolar basis) until reproducible responses were recorded. Concentration response experiments were made in a cumulative way with angiotensin II (Hypertensin’“‘. Ciba Geigy, Basel, Switzerland) by adding the drug in volumes exceeding not more than 0.3% of the tissue bath to reach final required concentration. Vessel sensitivity to angiotensin II was determined using iterative non-linear regression analysis, fitting the vessel response to the classical logistic function; R/R,,, = [A”/(A” +EC,,“)], where R/K,,,, is vessel A

I3 5 mN

5mN

C

l”i__

10 mN K+

A II

K+

A II

5 min FIG. 1. Upper tracings show the effect of cumulative addition of angiotensin II. lo- “--lO~j M, on isolated 1040 pm bovine posterior ciliary (A) and 22 5 pm retinal resistance artery (B). Addition of angiotensin II to the organ bath is indicated by dots and values show log (angiotensin II. ([MI). Lower tracings show effect of single 10e6 M angiotensin II, (A II) concentration, on isolated 653 ym bovine posterior ciliary artery (C) and 225 pm retinal resistance artery (D). K’ indicates change of bathing solution to 12 5 mM potassium solution, K-PSS. W is wash-out in normal PSS. Vertical scales show force in mN.

ANGIOTENSIN

II IN

RETINAL

471

ARTERIES

per metre vessel wall, N m-l), calculated as increase in vessel wall force (N) above resting level divided by twice the vessel segment length (m). Differences between means have been tested using Student’s t-test for paired and unpaired data where appropriate, with the level of significance set at P < 0.05 in either case. 3. Results Angiotensin II, 10-*“-10-6 M, induced a small transient concentration-dependent contraction in isolated bovine posterior ciliary arteries [Fig. l(A)], whereas no detectable contraction induced by angiotensin II, 10-lo-lO-s M, was observed in any of the retinal resistance arteries [Fig. l(B)]. Figure 2 shows the average concentration response curves of the retinal resistance arteries [internal lumen diameter, Zo, 2 3 5 + 45 ,um (S)] and posterior ciliary arteries (I, : 6 72 &- 8 6 pm ( 1O)]. The pD,-value for angiotensin II was 9.3 + 0.8, corresponding to an EC,, concentration Of 5 X 10-l’ M. The effect of a single high concentration of angiotensin II, 10m6M, on the retinal and ciliary arteries is shown in Fig. 1 (lower). The response of the posterior ciliary arteries [I, : 707+145/Jm (7)] to 10-6M angiotensin II was transient, and vessel wall tension had declined to resting level within 2-5 min [Fig. 1 (C)l. The maximal contraction of the posterior ciliary arteries increased significantly (P < 0.05) from 5 f 2 % (10) of maximal K-PSS induced contraction during cumulative angiotensin II activation, to 15 + 4 % (7) by single 10efi M dose stimulation. The 0.5

ciliary arteries were UnreSpOnSiVe to IO-’ M angiotensin II, except for one artery, when challenged again after up to 1 hr after wash-out of the first dose. Stimulation of the retinal resistance arteries [I,: 170 &- 11 ,um (S)] with a single dose of angiotensin II did not induce contraction in these vessels [Fig. l(D)]. Contraction of the retinal arteries to angiotensin II could not be elicited by blockade of the cyclooxygenase enzyme with indomethacin. lo-” M, with endothelium-dependent relaxing factor (EDRF)-induced cyclic guanyl formation blocked with 3 x 10m6 M methylene blue, and in vessels devoid of endothelium, verified by the absence of acetylcholine-induced relaxation of the arteries after they were precontracted by 10m5 M PGF,, (Furchgott and Zawadzki, 1980). Repetition of the angiotensin II concentration response curve on the posterior ciliary arteries demonstrated development of tachyphylaxis to angiotensin II (Fig. 2). Maximal response of vessels to angiotensin II during first stimulation was 0.44 & 0.06 N m-’ and 0.02 + O-02 N m-l (5) (P < 0.001, paired t-test) during last exposure. In order to study a possibly relaxing effect of angiotensin II, the retinal resistance vessels were precontracted with PSS containing 30 mM potassium, as these arteries do not have a spontaneous myogenic tone when kept in PSS. Angiotensin II did not induce relaxation of the vessels under this condition (Fig. 3). Initial contraction of the vessels to 30 mM potassium was 0.15+0.04 N m-l (5). Angiotensin II, 10m6M, did not potentiate the contraction of retinal resistance (Fig. 4, right) and posterior ciliary arteries (Fig. 4, left) induced by 5hydroxytryptamine and noradrenaline over the concentration ranges tested. Table I gives the morphological and mechanical data of the arteries.

0.4 T E z

0.3 1

- 10

-9

-8 log (Angiotensin

-7 II,

-6

[M])

FIG. 2. Angiotensin II concentration response characteristics of isolated bovine retinal (a) and posterior ciliary arteries exposed to A II first (m). and second (0) time. Responses are shown as active wall tension (Nm-I), measured as increase in vessel wall force above resting level divided by twice the vessel segment length. Points show mean of five retinal and ten posterior ciliary arteries, and five vessels and vertical bars show +s.E.M. Effective lumen diameter of vessels. I,,, was 172 +8 pm (5) and 672 + 86 Frn (lo), for the retinal and posterior ciliary arteries, respectively. Effective lumen diameter, I,, of posterior ciliary arteries exposed second time to A II was 699 + 122 pm (5).

I

-10

-9

-7

-0 log (Angiotensin

il.

-6

[M])

FIG. 3. Angiotensin II concentration response relation (0) of isolated retinal resistance arteries precontracted with PSS containing 30 IIIM potassium. Time control responses of vessels shown by open circles. Responses have been normalized to the vessel wall tension just before addition of the first A II concentration to the tissue bath. Points show mean response of five arteries. Vertical bars show +s.E.M. Effective lumen diameter, I,, was 240+ 12 /urn (5).

472

N. C. 8. NYBORG

-9

-8

-7

-6

ETAL

-5 loa (5-t-i~,

-8

-7

-6

-5

-4

FIG. 4. Effect of 1O-6 M angiotensin II (0) on the 5-hydroxytryptamine (S-HT) (upper) and noradrenaline (NA) (lower) concentration response characteristicsof eight isolatedbovine retinal resistancearteries(right) and six posteriorciliary arteries

(left). Responses are shownasactive wall tension(N/m), calculatedasincreasein vesselwall force aboverestinglevel divided by twice the vesselsegmentlength. Pointsshowmeanand vertical bars + S.E.M. Vesselcharacteristicsand mechanicaldata are shownin Table 1. Note the differencein vertical scalebetweenretinal and posteriorciliary arteries.

TABLE

I

Vesseldimensionsand mechanicaldatafor efSectof 10 -6 M angiotensinI1 (A 11)on 5hydroxytryptamine (5HT) and noradrenaline(NA) concentration responsecharacteristics of isolated bovine retinal resistanceand posterior ciliary arteries. Control Retinal resistance arteries AT-S-HT (N m-i) 0~20~0~05 (8) AT-NA (N mm’) 0.09 * 0.02 17Okll I, (w-d Posterior ciliary arteries AT-5-HT (N m-l) 10.02 k 5.00 (6) AT-NA (N mm’) lO.llf2.99 636&153 1, (,m)

+A

II

0.16+0.05 0~14~0~05 (8)

P

(8)

N.S. N.S.

10.43 k4.26 (6) 10.74* 3.14 (6)

N.S. N.S.

Values given as mean + S.E.M. (number of vessels). AT-5-HT and AT-NA is maximal active well tension (N m-l) developed by the vessels during 5-HT and NA stimulation, respectively. I, is effective lumen diameter of vessels (see Materials and Methods). P is probability for difference between mean values tested by Student’s ttest for paired data. N.S. is not significant.

4. Discussion Angiotensin II is an important regulator of several physiological systems such as blood pressure and

intravascular volume (Re, 1984). The action of angiotensin II on the ocular circulation is sparingly studied (Dollery et al., 1963 ; Alm, 1972 ; Rhie et al., 1982 ; Rockwood et al., 198 7) exclusively by means of injection of angiotensin II in vivo. Angiotensin II has, in this way, been reported to have constrictory effect or no effect at all on the retinal vasculature and blood flow. It is, however, a matter of debate whether the effect of angiotensin II in vivo is due to direct activation of the retinal vasculature or to indirect activation through autoregulatory mechanisms in the ocular vascular bed, because of interference with blood flow and pressure outside the retinal circulation. Evidence for a direct action of angiotensin II on the retinal vasculature has only been provided after intravitreous injection in cats (Rockwood et al., 1987), which may indicate that species-dependent variability in retinal angiotensin reactivity may exist. Both the choroidal and retinal vasculature appear capable of forming angiotensin II, since the ACE is present in this tissue (Ward et al., 1979 ; Igic and Kojovic, 1980; Ferrari-Dileo et al., 1988). Angiotensin II receptors have also been detected in both human and bovine retinal vessels (both arteries and veins) using specific radio-ligands (Ferrari-Dileo et al., 1987),

ANGIOTENSIN

II IN RETINAL

473

ARTERIES

suggesting that these vessels may be sensitive to angiotensin II. The retinal vascular angiotensin II receptor resembled angiotensin II receptors in other tissues with respect to the influence of guanine nucleotides and cations (Mg2+ and Na’) on the binding site characteristics. However, the retinal angiotensin II-receptor density, 0.8 fmol mg-’ protein, appears to be strikingly low compared with other types of tissues : approximately 22 fmoi mg-’ protein in dog cerebral microvessels (Speth and Harik, 1985) and 245 fmol mg-l protein and 720 fmol mg-’ protein for high and low affinity binding sites, respectively, in cultured myocytes (Rogers, Gaa and Allen, 1985). It seems therefore to be at least a 25-fold difference in receptor density between the retinal and cerebral vessel preparation. The present experiments demonstrate a striking lack of effect of angiotensin II on the bovine retinal resistance arteries. Angiotensin II was, in contrast, a potent, but not very effective vasoconstrictor in the posterior ciliary arteries. A likely explanation for this discrepancy may be related to receptor density, or loss of function of angiotensin II receptor in terms of vascular smooth muscle force generation, when the posterior ciliary artery branches and forms the retinal circulation. The passive mechanical properties of the retinal resistance arteries may also possibly absorb a very weak contraction of the vascular smooth muscle cells (Mulvany and Warshaw, 1979). The potentiating effects on other receptor-evoked vascular responses, e.g. on 5-hydroxytryptamine, and exogenous as well as endogenous neuronal noradrenaline induced vessel contractions, is a wellknown action of angiotensin II (Peach, 19 77). In our experiments we could not find such effect of angiotensin II either on the retinal resistance or on the posterior ciliary arteries, even though the latter arteries did respond to angiotensin II. This difference might be related to variation in animal species or angiotensin II receptor function. Since both the enzymatic and receptor system for angiotensin II is present in bovine retinal vessels it is surprising to find that the retinal vessels used in our experiments were insensitive to angiotensin II. It often appears difficult to show a direct contractile effect of angiotensin II on vascular tissue in vitro and when possible. tachyphylaxis is then commonly encountered (Peach. 1977). The vascular endotheiium seems to augment the development of tachyphylaxis (Gruetter, Ryan and Schoepp. 1987). Experimentally this was checked by activating the vessels with one single high angiotensin II concentration and removing the endothelium, but without effect in the retinal resistance arteries. The response of the posterior ciliary arteries to angiotensin II, in which tachyphylaxis was pronounced, was tripled by single dose stimulation. We did not study the influence of endothelium on the development of tachyphylaxis in these vessels. We cannot, however, exclude the possibility that the

renin-angiotensin system has been fully activated during destruction of the animals, and thus may have caused inactivation of the angiotensin II receptors in the retinal resistance arteries before we could test the vessels. One major problem with radio-ligand binding techniques is that information about the physiological response coupled to the receptor studied will not be determined. The angiotensin II receptors are not necessarily coupled to vascular smooth muscle contraction. Angiotensin II has been shown to cause contraction of aortic endothelium (Robertson and Khairallah, 19 72), increase brain capillary permeability after intraventricular injection, and also to relax vascular tissue (Toda, 1972). However, angiotensin II did not induce relaxation in the retinal arteries when these were precontracted with 30 mM potassium. In conclusion, we have shown that bovine arteries supplying eye structures are distinct in their reaction to angiotensin II. Retinal resistance arteries do not react in vitro to angiotensin II. and neither is the effect of 5-hydroxytryptamine and noradrenaline potentiated, suggesting that the number of angiotensin II receptors may be too small to affect the retinal vascular smooth muscle tone, or that the receptors are unrelated to the contractile system of the vascular smooth muscle cells. The present method, however, does not exclude the possibility that angiotensin II may have an effect in segments of the bovine retinal vascular bed more distal to those studied here. Acknowledgements This work was supportedby The P. Carl PetersenFoundation, DanskBlindesamfund,and DanishMedicalResearch Council Grant No. 12-8717. We are grateful for the eyes donatedby Aarhus OffentligeSlagtehus. References Aim, A. (1972). Etrects of norepinephrine, angiotensin, dihydroergotamine, papaverine, isoproterenol, histamine.nicotinic acid, andxanthinol nicotinateon retinal oxygen tension in cats. Acta Ophthalmologica 50, 707-19.

Dollery, C. T.. Hill, D. W. and Hodge, J. V. (1963). The responseof normal retinal blood vesselsto angiotensin and noradrenaline.I. Physiol. (Lund.) 165, 500-7. Ferrari-Dileo,G., Davis, E.B. and Anderson, R. D. (1987). Angiotensinbinding sitesin bovine and human retinal blood vessels. Invest. Ophthalmol. Vis. Sci. 28. 1747-51.

Ferrari-Dileo,G., Ryan, J. W., Rockwood,E.J.. Davis, E.B. and Anderson, D. R. (1988). Angiotensin-converting enzyme in bovine, feline and human ocular tissue. Invest. Ophthalmol. Vis. Sci. 29, 876-81. Furchgott, R. F. and Zawadzki, J. V. (1980). The obligatory role of endothelial cell in the relaxation of arterial

smoothmuscleby acetylcholine.Nature398, 373-6. Gruetter. C. A.. Ryan, E. T. and Schoepp. D. D. (1987). Endothelium enhances tachyphylaxis to angiotensins II and III in rat aorta. Eur. 1. Pharmacol. 143, 13942. Igic, R. P. and Kojovic. V. (1980). Angiotensin I converting

474 enzyme (kinase II) in ocular tissue. Erp. Eye Res. 30, 299-303. Mulvany. M. J. and Halpern. W. (1976). Contractileproperties of small arterial vesselsin spontaneouslyhypertensiveand normotensiverats. Circ. Res. 41, 19-26. Mulvany. M. J. andNyborg, N. ( 1Y80). An increasedcalcium sensitivity of mesentericresistancevesselsin spontaneously hypertensive rats. Rr. 1. Pharnlacol.71, 585-96. Mulvany, M. J. and Warshaw.D. (19 79). The active tensionlength curve of vascular smoothmusclerelated to its cellular components.1. Gen.Phgsiol. 74, 85-104. Nielsen,P. J. and Nyborg. N. C. B. (1989a). Calciumantagonist-induced relaxation of the prostaglandin-F,, responseof isolatedcalf retinal resistancearteries. Exp. Eye Res.48, 329-35. Nielsen, P. J. and Nyborg. N. C. B. (1989b). Adrenergic responses in isolatedbovine retinal resistancearteries. lnt. Ophthalmol.13. 103-7. Nyborg, N. C. B.. Baandrup, U., Mikkelsen, E.0. and Mulvany, M. J. (1988). Active, passive and myogenic characteristics of isolated rat intramural coronary resistancearteries.PfliigersArchiv/Eur. 1. Physiol. 410, 644-70. Peach, M. J. (1977). Renin-angiotensinsystem: Biochemistry and mechanism of action. Physiol. Rev. 57, 313-70. Re, R. N. (1984). Cellular biology of the renin-angiotensin systems.Arch. Intern. Med. 144, 2037-41.

N. C. B. NYBORG

ETAL

Rhie. F. H.. Christlieb. A. K.. Sandor. T.. (Gleason.K. E., Rand. L. 1.. Shah. S. T. and Soeldner.J. S. (1982). Retinal vascular reactivity to norepinephrine and angiotensin-II in normals and diabetics.Dinhrtes31, 1056-60. Robertson,A. L. and Khairallah. P. A. ( 1972). Effectsof angiotensin II and someanalogueson vascular permeability in the rabbit. Circ. Res. 31, 92 3-3 I. Rockwood,E.J., Fantes,F.. Davis,E.B. and Anderson,D. R. ( 1987). The responseof retinal vasculature to angiotensin. Invest. Ophthalmol.Vis. Sci. 28, 676-82. Rogers.T. B., Gaa.S.T. and Allen, I. S.(198 5). Identification and characterization of functional angiotensinII receptorson cultured heart myocytes. 1. Pharnmo~. Exp. Ther. 236, 438-44.

Sossi,N. and Anderson, D. R. (1982). Blockageof axonal transportin optic nerve inducedelevationof intraocular pressure.Arch. Ophthalmol.101, 94-7. Speth.R. C. and Harik, S. I. ( 1985). Angiotensin II receptor bindingsitesin brain microvessels. Prur. Nafl. ifcud. Sri. U.S.A. 82. 6340-3. Toda, N. (1972). Mechanism of vascular smooth muscle relaxation inducedby angiotensins.In Vasodilation (Eds Vanhoutte. P. M. and Leusen.I.). Pp. 151-7. Raven Press:New York. Ward, P. E., Stewart, T. A., Hammon.K. J., Reynolds.R. C. andIgic. R. P. (1979). AngiotensinI converting enzyme (KinaseII) in isolatedretinal microvessels. L$eSci. 24, 1419-24.