Lack of specific (3H) prazosin binding sites in dog and rabbit cerebral arteries

Life Sciences, Vol. 35, pp. 2169-2176 Printed in the U.S.A.

Pergamon Press

LACK OF SPECIFIC (3H) PRAZOSIN BINDING SI~ES IN DOG AND RABBIT CEREBRAL ARTERIES Patrice M. Ferron, William Banner, Jr. and Sue Piper Duckles Departments of Pharmacology and Pediatrics College of Medicine University of Arizona Tucson, Arizona 85724 (Received in final form September i0, 1984) SUMMARY In order to explore the characteristics of alpha adrenergic receptors on cerebrovascular smooth3~uscle , specific binding sites for the alpha I adrenergic ligand, ( H ) p~azosin, were studied in blood vessel homogenates. No specific ( H ) prazosin binding was found in either rabbit or dog cerebral arteries, but specific binding was demonstrated in the rabbit saphenous and ear arteries. In the ear artery H-prazosin binding was saturable with a K d of 0.51 ± 0.20 nM and a Bmax of 89 ± 29 fmo les/mg protein. To confirm the adequacy of our membrane preparation, homogenates of both dog and rabbit cerebral arteries showed saturable specific bindin~ with two different ligands: one for muscarinic receptors, [ HI(-) quinuclidiny~ benzilate (QNB) and one for alpha 2 adrenergic receptors, ( h ) yohimbine. The results of these studies demonstrate a lack of alpha I adrenergic receptors on cerebral blood vessels, confirming functional studies showing only a weak contractile response to norepinephrine. The control of cerebral blood flow has long been an issue of considerable controversy. Cerebral blood flow may be regulated by four major factors: chemical stimuli (blood gases and pH), autoregulation, metabolic factors and neural stimuli (I). Of these four influences on the cerebral circulation, perhaps the least understood is the role of neural stimuli in the regulation of cerebral blood flow. Fluorescence histology, electron microscopy and studies of transmitter content, release and accumulation have demonstrated the presence of a dense adrenergic nerve supply to the cerebmal vasculature (2,3,4,5,~). Despite this evidence for ample innervation, cere~ral arteries exhibit only minimal contractile responses to stimulation of sympathetic nerves im vitro (7,8,9). This limited response to transmural~nerve stimulation can be am~ounted for by the weak responsiveness of cerebrovascular smooth muscle to exogenous norepinephrine (10,11,12,13). A~lous properties of cerebrovascular alpha adrenergic receptors have b e e n ~ o t e d by many investigators including weak responsiveness of cat cerebral arteries to phenylephrine, an alpha-adrenergic agonist (I0) and a lack of contractile responses of the rabbit basilar artery to imidazoline derivatives which do cause contraction of the pulmonary artery (14). Recently contractile responses of feline cerebral arteries have been shown to be mediated primarily by alpha 2 adrenergic receptors (15,16). 0024-3205/84 $3.00 + .00 Copyright (c) 1984 Pergamon Press Ltd.

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Because of the unique properties of cerebrovascular responses to adrenergic stimulation, we wondered whether lack of responsiveness to alpha 1 adrenergic agonists and antagonists of dog and rabbit cerebral blood vessels is due to a lack of receptors. Therefore, using radio-ligand binding t~chniques, we have investigated the characteristics of specific binding of ( H ) prazosin, a ligand for alpha I adrenergic receptors (17,18) in homogenates of dog and rabbit cerebral arteries. METHODS Tissue Preparations New Zealand white rabbits (203 kg) of either sex were killed by decapitation. Mongrel dogs (15-40 kg) of either sex were killed by injection of 2-3 ml/kg of thiopental (7.5 mg/ml) and barbital (125 mg/ml). Brains were removed and placed in physiological saline at room temperature. The cerebral blood vessels were dissected from the surface, including the anterior, middle and posterior cerebral arteries, the basilar artery and the circle of Willis. The ear artery and saphenous artery were also removed from the rabbits. Vessels were frozen at -15°C until 100-200 mg of tissue could be accumulated (i-2 weeks), after it had been determined in the rabbit aorta that no deterioration of radioligand binding occurred in this time period. In one experiment fresh aorta and aorta frozen for two weeks were compared giving K d values of 0.79 and 1.4 nM and Bmax values of 53 and 81 fmoles/mg protein respectively. This confirms previous reports that freezing tissue does not alter the binding characteristics (19). On the day of assay, the tissue was thawed, mixed with 6 ml of assay buffer (see below) and homogenized at setting O on a Polytron (Brinkmann Instruments) for 30 sec. The homogenate was centrifuged at 3000 x g for 15 min, and the pellet discarded. The supernatant was transferred to nitrocellulose tubes and centrifuged at 120,O00 x g for one hr at 5°C. The supernatant was discarded and the pellet resuspended at Polytron setting 6 for 15 sec in assay buffer to produce a concentration of I-2 mg of protein/ml as measured by a Lowry assay (20). (3H) Prazosin Bindin~ Furoyl-5-13H]prazosin was obtained from New England Nuclear (NEN) (specific activity 17.1 Ci/mmol). 3Tissue homogenate (200 ~i) was incubated with different concentrations of ( H)prazosin for 30 min at 23°C in a final volume of 400 ~i of 50 mM Tris NCI with i mM MgCI 2 (pH 7.5). Non-spec_i~ic binding was defined as radioactivity bound in the presence of 2.5 x I0 M lnorepinephrine (Sigma Chemical Company). The incubation was terminated within 10 sec by addition of 3 ml of ice cold assay buffer and filtration through a GF/C glass fiber filter (Whatman) in a vacuum filtering apparatus designed to filter at 2.5 ml/sec. The filter was rinsed three times with 3 ml of ice cold buffer. (3H) prazosin remaining on the filter was extracted for 16 hr with 8 ml of scintillation fluid [2:1 toluene:triton X-100 (NEN) and 16 g/l Omnifluor (NEN). Radioactivity was determined in a Beckman LS-230 unrefrigerated liquid scintillation counter (40% efficiency). [3H](-)qNB Bindin~ [3H](-)quinuclidinyl benzilate, L-[benzilic-4,4'-3H] (QNB) was obtained from New England Nuclear (specific activity 33.1Ci/mmol) (21). Tissue homogenate (200 ~i) was incubated with various concentrations of [3H](-)QNB for 60 min at 37°C in a final volume of 400 ~i of 50 mM Na/K phosphate buffer (pH 7.4). Non-specific binding was defined as radioactivity bound in the

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[3H] prazosin Binding in Cerebral Arteries

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presence of 2.5 x 10 -7 M atropine (Sigma Chemical Co.). All other procedures were identical to those listed above. (3H) Yohimbine Binding Yohimbine [methyl-3H] was obtained from New England Nuclear (specific activity 81.2 Ci/mmol). Tissue homogenate (200 ~i) was incubated with different concentrations of ( H ) yohimbine for 30 min at 23°C in a final volume of 400 ~i of 50 mM Tris, 0.5 mM EDTA, 0.;% ascorbic acid, pH 7.5. Nonspecific binding was defined as radioactivity bound in the presence of 2.5 x I0 M phentolamlne HCI (Ciba Geigy Corp.). All other procedures were identical to those listed above. Data Analysis Data analysis was performed using nonlinear regression on the raw data counts using equations I and 2. Nonspecific Binding = M.L

(I)

Total Binding = (M.L) + [Bmax

A.

(I + Kd/L)]

(2)

RABBIT CEREBRAL ARTERIES 100-

,,-, Z

80.

0 = z

~

~o.

"~" O E

4.0"

U)

o N < ¢ Z ~

20.

0

FREE ( s H ) P R A Z O S I N (nM)

R A B B I T EAR A R T E R Y

B.

1000 Z

0

=

80-

z

~ ~,

N

~

60"

4-0"r v

20-

0

~

~

~

FREE ( 3 H ) P R A Z O S I N (nM)

Figure I: (3H) Prazosin binding in homogenates of rabbit cerebral arteries (A) and rabbit ear artery (B). Total and nonspecific binding (fmoles bound/mg protein) are plotte~ as a function of free (3H) prazosin concentration (means and Total S.E., N=3 for cerebral A A arteries; a single experiment done in Non-specific duplicate is illustrated X---X for ear artery).

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[3H] Prazosin Binding in Cerebral Arteries

M is the slope of t ~ line of concentration for nonspecific sites, K d is the dissociation Values are presented as means

Vol. 35, No. 21, 1984

femtomoles bound/mg protein vs. ligand binding, Bmax is the maximal number of binding constant and L is the ligand concentration. ± S.E. RESULTS

As can he seen in Figure IA, there are no specific binding sites for (3H) prazosin in homQgenates of rabbit cerebral arteries. In other words, curves for total and non-specific binding are superimposable. Similar results were found in homogenates of dog cerebral arteries (Table i). This lack of specific binding s~tes was not due to storage of the tissue at -20°C as use of fresh tissue did not give different results. In one experimeqt dog cerebral arteries that ~ad not been frozen were used, and no specific ~H prazosin binding was found.

TABLE 1 (3~) Prazosin Specific 5knding in Cerebral Arteries of the D~g and Rabb~t and Rabbit Peripheral Arteries

Species

Rabbit Dog Rabbit Rabbit

~ssue

Cerebral ArTeries Cerebral Arteries Ear Artery Saphenous Artery

K d (nM)

--0.51 ± 0.20 0.37 ± 0.I0

Bmax (fmoles/mg protein)

0 0 89 ± 29 91 ± 45

N

4 3 2 2

In contrast, in the rabbit ear artery ~here was a significant difference between total and n~nspecific binding of ( H ) prazosin (Fig. IB). In the rabbit ear artery, (JH) prazosin binding was saturable with a K d of 0.51 ± 0.20 nM and a Bmax of 89 ± 29 fmoles/mg protein. Similar results were obtained in the rabbit saphenous artery (Table I). In contrast to the lack of (3H) prazosin binding in cerebral arteries, we re able to demonstrate specific binding of the alpha 2 adrenergic ligand, ) yohimblne, in rabbit (n=3) and dog (n=l) cerebral arteries (Fig. 2).

TABLE 2 [3H](-)QNB Specific Binding in Rabbit and Dog Cerebral Arteries

Species

Rabbit Dog

Tissue

K d (nM)

Cerebral Arteries Cerebral Arteries

0.062 ± 0.05 ND

ND = Not determined.

Bmax (fmoles/mg protein)

79 ± 9 89 ± 8

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[3H] Prazosin Binding in Cerebral Arteries

RABBIT

CEREBRAL

ARTERIES

200Total Z~ Z~

C3 Z

O ~

Non-specific X---X

150-

Z

~

o

o

E

"1"

FREE

[=HIYOHIMBINE(nM)

Figure 2: [3H]Yohimblne binding in homogenate of rabbit cerebral arteries. Total and non-specific binding (fmoles bound/mg protein) are plotted as a function of free (3H)yohlmbine concentration for a single experiment done in duplicate. Kd=6.7 nM; Bmax=102 fmoles/mg protein.

RABBIT

CEREBRAL

ARTERIES

150-

Total A a Z

~

i

,-~

E ~

Non-specific X---X

f 50-

-r

0 ,

o'.2

o'.,

0'.6

o'.s

i

FREE [ 3 H ] ( - ) Q N B ( n M )

Figure 3: [3H](-)QNB binding in homogenates of rabbit cerebral arteries. Total and non-specific b~nding (fmoles/mg protein) are plotted as a function of free [JH](-)QNB concentration. A single experiment done in duplicate is shown as an example.

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[3H] Prazosin Binding in Cerebral Arteries

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To further confirm the appropriateness and adequacy of our membrane preparation, another ligand, ( H)-QNB, which is specific for muscarinic binding sites, was used. As can be seen in Figure 3, there was a significant difference between total and non-specific (~H) ~NB binding in homogenates of cerebral arteries. This binding was saturable with a K d o~ 0.062 ± 0.05 nM and Bmax of 79 ± 9 fmoles/mg protein (Table 2). Specific [JH](-)QNB binding was also seen in dog cerebral arteries (Table 2). DISCUSSION One of our primary concerns after finding no specific (3H) prazosin binding in cerebral arteries was that this was an artifact of our membrane preparation. We therefore examined other tissues and used two different r~dioligands to show that this was not the case. We feel that the lack of ( H ) prazosin binding does in fact correlate with a lack of alpha I adrenergic receptors on cerebral vessels for the following reasons: I. There was no specific (3~) prazosin binding in cerebral arteries of two species (rabbit and dog). Cerebral arteries from these two species have different structural properties and contain differing amounts of elastic ~ d connective tissue. Thus it does not seem likely that the lack of specific ( H ) prazosin binding sites could be due to problems of the homogenization procedure specific to cerebral vessels. 2. Specific (3H) prazosin binding could be demonstrated in vessels known to respond to alpha I adrenergic stimulation. Using methods identical to those used for cerebral arteries specific ( H ) ~razosin binding was demonstrated in the rabbit ear and saphenous arteries. ( H ) prazosin binding has also been demonstrated in the aorta and femoral, renal and mesenteric arteries of the dog, with a K d of 0.2 nM (22). This is similar to the K d values we found in the rabbit ear and saphenous arteries (0.51 ± 0.20 and 0.37 ± 0.I0 nM, respectively). 3. In contrast to the lack ot specific binding with an alpha 1 adrenergi~ ligand, we did find specific binding of an alpha 2 adrenergic ligand, ( H ) yohimhine, in homogenates of both rabbit and dog cerebral arteries. This suggests that although alpha I adrenergic receptors are absent, there are alpha 2 adrenergic receptors present. 4. Using a radioligand which is specifi~ for another class of membrane bound binding sites, we have shown specific [ N](-)QNB binding in homogenates of cerebral arteries. This further supports the adequacy of our membrane preparation. Specific [JH](-)QNB binding has also been demonstrated in bovine pial arteries (apparent Kd, 0.54 nM; Bmax, 693 fmoles/mg protein) as well as in bovine cerebral microvessels (23). We have also demonstrated specific [3H](-)QNB binding in other rabbit blood vessels: the aorta and ear artery (unpublished observations). The presence of muscarinic binding sites in the wall of cerebral blood vessels corresponds to the well-established ability of these vessels to relax to low concentrations of acetylcholine and contract in response to high concentrations (24). 5. Although it cannot be assumed that binding sites are equivalent to receptors, our findings of a lack of alpha I adrenergic binding sites in cerebral arteries do correlate with pharmacological evidence of unusual properties of cerebrovascular contractile responses to norepinephrine. In many species, including the dog, cat and rabbit, contractile responses of cerebral arteries to norepinephrine are relatively small and large concentrations are required to produce a response (9,10,11). This weak responsiveness of dog cerebral arteries has been confirmed using

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electrophysiological techniques (12). The rat basilar artery does not contract at all to norepinephrine (13) and the lack of alpha adrenoceptor b~ing sites in this tissue has been confirmed using the radioligand ( I)BE2254 (25). Recently using specific agonists and antagonists for the two classes of alpha adrenergic receptor, contractile responses of the isolated feline middle cerebral artery have been shown to be mediated predominantly by the alpha 2 class of receptor (15,16). The unusual complement of alpha adrenergic receptors in cerebrovascular smooth muscle may in part account for the weak responsiveness to adrenergic nerve stimulation in the face of much evidence for a dense and functionally active innervation. In most blood vessels, it has been hypothesized that norepinephrine released from nerves causes smooth muscle contraction by an action on alpha I adrenergic receptors (26). In rabbit and dog cerebral blood vessels, adrenergic nerve stimulation produces a weak contractile response that is not blocked by ~-adrenergic antagonists (7,9) correlating with the lack of alpha I adrenergic receptors. The weak neurogenic contraction of cerebral blood vessels could be accounted for by an action on a unique class of adrenergic receptors (27) or by the actions of a co-transmitter, such as neuropeptide Y (28). At any rate, the unique complement of cerebrovascular adrenergic receptors needs to be taken into account when exploring the role of adrenergic innervation of this vascular bed (29). ACKNOWLEDGEMENTS Our grateful thanks to Dr. Henry I. Yamamura for advice and support in this work. Supported in part by NSF Grant #BNS 79-24750 and NIH grant HL30956, and by a Grant-in-Aid from the Arizona Affiliate of the American Heart Association. SPD is an Established Investigator of the American Heart Association with funds provided in part by the Arizona Affiliate. PMF was supported by a medical student research fellowship, NIH grant ~T35HLO7479. REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9. I0. II. 12. 13. 14. 15. lb. 17. 18. 19.

D. D. HEISTAD and M. L. MARCUS, Circ. Res. 42 295-302 (1978). T. IWAYAMA, J. B. FURNESS, and G. BURNSTOCK, Circ. Res. 26 635-64b (1970) S.P. D U C K L E S and R. RAPOPORT, J. Pharmacol. Exp. Ther. 2! 1 219-224 (1979). S. P. DUCKLES, Brit. J. Pharmacol. 69 193-19~ (1980). T . J . F . LEE, C. C. CHUIEH and M. ADAMS, Eur. J. P h a r m a c o l . 61 55-70 (1980). T. J. F. LEE, Circ. Res. 49 971-979 (1981). T. J. F. LEE, C. SU and J. A. BEVAN, Circ. Res. 39 120-126 (1976). I. MURAMATSU, M. FUJIWARA, Y. OSUMI and S. SHIBATA, Blood Vessels 15 II0118 (1978). S. P. DUCKLES, Circ. Res. 44 482-490 (1979). L. EDVINSSON, and C. OWMAN, Circ. Res. 35 835-849 (1974). S. P. DUCKLES and J. A. BEVAN, J. Pharmacol. Exp. Ther. 197 371-378 (1976) S. FUJIWARA, T. ITOH and H, SUZUKI, Brit. J. Pharmacol. 77 197-208 (1982). R. J. WINQUIST and D. F. BOHR, Experientia 38 1187-1188 (1982). J. A. BEVAN, J. Pharmacol. Exp. Ther. 216 83-89 (1981). T. SKARBY, K. E. A N D E R S O N and L. E D V I N S S O N , Acta Physiol. Scand. 112 105107 (1981). I. C. MEDGETT and S. Z. LANGER, N-S Arch Pharmacol. 323 24-32 (1983). P. GREENGRASS and R. BREMNER, Eur. J. Pharmacol. 55 323-326 (1979). R. HORNUNG, P. PRESEK and H. GLOSSMANN, N-S Arch. Pharmacol. 308 223-230 (1979). A. SASTRE, K. K. GRIENDLING, M. M. R U S H E R and W.R. MILNOR, J. Pharm. Exp.

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20. 21. 22. 23. 24. 25. 20.

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Ther. 229, 887-896 (1984). O. H. LOWRY, N. J. ROSEBROUGH, A. L. FARR and R. J. RANDALL, J. Biol. Chem. 193 265-275 (1951). H. I. Y A M A M U R A and S. H. SNYDER, Proc. Nat. Acad. Scl. USA 71 1725-1729 (1974). A. BOBIK, Life Sciences 30 219-228 (1982). C. ESTRADA and D. N. KRAUSE, J. Pharmacol. Exp. Ther. 221 85-90 (1982). T. J. F. LEE, Eur. J. Pharmacol. 68 393-394 (1980). G.D. S I L V E R B E R G , T. O. NEILD, A. A D A M S and B. JARROTT, N e u r o s c i e n c e Letters 32 109-111 (1982). I. Y A M A G U C H I and I. J. KOPIN, J. Pharmacol. Exp. Ther. 214 275-281

(t980). 27. 28. 29.

G. D. S. HIRST and T, O. NEILD, J. Physiol. 313 343-350 (1981). L. EDVINSSON, P. EMSON, J. MCCULLOCH, K. T A T E M O T O and R. UDDMAN, Neurosci. Left. 43 79-84 (1983). S.F. VATNER, L. L. PRIANO, J. D. R U T H E R F O R D and W, T. MANDERS, Amer. J. Physiol. 238 H594-H598 (1980).