Arginine vasopressin receptors in pig cerebral microvessels, cerebral cortex and hippocampus

Neuroscience Letters, 87 (I 988) 121-126 Elsevier Scientific Publishers Ireland Ltd.

121

NSL 05245

Arginine vasopressin receptors in pig cerebral microvessels, cerebral cortex and hippocampus A. Frances Pearlmutter l, Mary Szkrybalo l, Younghee Kim 1 and S a m i I. H a r i k 2 1Department of Biochemistry, Medical College of Ohio, Toledo, OH 43699 (U.S.A.) and 2Department of Neurology and Pharmacology, Case Western Reserve University School of Medicine, Cleveland, OH 44106 (U.S.A.)

(Received 30 October 1987; Revised version received 21 December 1987; Accepted 23 December 1987) Key words." Arginine vasopressin receptor; Brain microvessel; Blood-brain barrier; Neural control of the brain microcirculation

We tested for the presence of arginine vasopressin (AVP) receptors in pig cerebral microvessels, cerebral cortex and hippocampus by specific binding methods with [3H]AVP as the ligand. The specific binding of [3H]AVP to all preparations was saturable and Scatchard analysis indicated a single class of high affinity binding sites (dissociation constant of 1-2 nM). Maximal binding capacity in cerebral microvessels was about 60% that of the cerebral cortex; and there were no apparent differences in the maximal binding capacity between cerebral cortex and hippocampus. These findings suggest the existence of AVP receptor sites in cerebral microvessels and support the hypothesis that AVP has a role in the control of the brain microcirculation.

Despite m u c h a n a t o m i c a l evidence s h o w i n g nerve fibers in close p r o x i m i t y to b r a i n b l o o d vessels, only recently are the functional significance a n d the identity o f the n e u r o t r a n s m i t t e r s being unraveled. I n n e r v a t i o n o f arteries a n d arterioles can m o d u late vascular resistance a n d c e r e b r a l b l o o d flow, while t h a t o f microvessels m a y regulate c a p i l l a r y r e c r u i t m e n t a n d b l o o d - b r a i n b a r r i e r (BBB) functions. A r g i n i n e vasopressin ( A V P ) is a p u t a t i v e p e p t i d e n e u r o t r a n s m i t t e r which can alter the p e r m e a b i l i t y o f b r a i n capillaries to n u t r i e n t substances [2, 6, 21] a n d w a t e r [5, 20]. A V P also affects several targets which include renal medulla, t o a d b l a d d e r , a o r t i c s m o o t h muscle, platelets, hepatocytes, b r a i n a n d a n t e r i o r p i t u i t a r y [14]. Studies o f the m e c h a n i s m s o f action o f A V P on these targets yielded results f r o m which a p a t t e r n for two m a j o r types o f A V P r e c e p t o r s has emerged: the h e p a t o c y t e receptor, linked to p o l y p h o s p h o i n o s i d e t u r n o v e r , was d e s i g n a t e d the V F t y p e receptor, while the renal m e d u l l a r y receptor, which is linked to cyclic A M P g e n e r a t i o n via a c t i v a t i o n o f a d e n y l a t e cy-

Correspondence." S.I. Harik, Department of Neurology, University Hospitals of Cleveland, Cleveland, OH 44106, U.S.A.

0304-3940/88/$ 03.50 O 1988 Elsevier Scientific Publishers Ireland Ltd.

122 clase, was designated the V2-type receptor [17]. Vl-type receptors were later described in vascular smooth muscle where they may be linked to Ca 2+ fluxes associated with muscle contraction [4]. The demonstration by ultrastructural immunohistochemical methods of the innervation of brain microvessels by AVP immunoreactive fibers [15], and the effects of AVP on the permeability of brain microvessels [2, 5, 6, 20, 21] led us to hypothesize that AVP receptors exist on brain microvessels. To test this hypothesis, we used radiolabeled ligand binding methods, previously used by one of us [3, 18], to assess and characterize AVP receptors in pig cerebral microvessels. In this communication, we describe the existence of specific, high affinity AVP binding sites in pig cerebral microvessels and in pig brain preparations. Binding characteristics of this receptor had structure-function relationships that resemble the Vwtype receptor. Brains of adult pigs were collected immediately after exsanguination at a local slaughterhouse. Cerebral cortical mantles and hippocampi were freed of meninges and choroid plexus. Microvessels were isolated from cerebral cortical mantles by bulk separation and their purity was assessed by microscopy and enzymatically [8, 9]. Microvessels were frozen at - 70°C until used for AVP receptor assays when they were thawed and washed in 50 mM Tris-maleate buffer, pH 7.4, with proteolytic inhibitors [18]. Microvessels were then homogenized in a ground glass homogenizer and the homogenates were centrifuged at 49,000g for 15 min. The supernatants were discarded and the pellets, which represent total particulate fractions, were washed and used for AVP receptor assays. All procedures were performed at (~4c'C unless otherwise stated. In some experiments, pig cerebral cortex and hippocampus were homogenized in ground glass homogenizers in 50 mM Tris-maleate buffer with proteolytic inhibitors [18], and the homogenates were centrifuged at 49,000 g for 15 min to obtain total particulate fractions, as described above for microvessels. In other experiments, brain tissues were homogenized in glass-teflon homogenizers in the Tris-maleate buffer, aE -0

60 --

Z

40

E

Cortex

/ i

o

~. 20 (~

sels

0

-10

I

I

-9

-8

log AVP [ M ]

Fig. 1. Saturation plots of specific[3H]AVPbinding to total particulate fractions of cerebral microvessels and cerebralcortex. Incubationconditionsare describedin the text.

123 now containing 0.32 M sucrose and proteolytic inhibitors. Homogenates were centrifuged at 1000g for 10 min, the pellets discarded and the supernatants centrifuged at 12,000g for 20 min to yield the P2 'crude synaptosomal' pellet. The supernatant was centrifuged at 160,000g for 45 min to get the P3 'crude microsomal' pellet. All pellets were resuspended in the receptor binding buffer described below. Protein was determined according to Bradford [1], with ovalbumin as standard. Tissue preparations (,-- 200/~g protein in 150 pl) were incubated in 50 m M Trismaleate buffer, p H 7.6, with 5 m M Ni 2+, Mn 2+ or Mg 2+ and 0.1% gelatin. [3H]AVP (87 Ci/mmol, [phenylalanyl-3,4,5-3H]AVP, New England Nuclear) was added at concentrations ranging from 0.1 to 10 nM. The final assay volume was 200/,1. Incubation took place for 1 h at 22°C with or without unlabeled AVP (5/IM) or one of its analogs, and was terminated by filtration under reduced pressure through G F / F W h a t m a n filters. The filters were rinsed with 15 ml of ice-cold buffer and their radioactive content determined by liquid scintillation at an efficiency of ~ 40 %. Specific binding was derived by subtracting the counts bound in the presence of 5 / t M unlabeled AVP from counts bound in its absence. Depending on the metal ion present and the concentration of [3H]AVP (0.1-10 nM), non-specific binding ranged from 50 to 80 % of the total binding. In all preparations, little or no specific binding could be detected in the absence of metal ion or with 5 m M Mg 2+ or Mn 2+. In the presence of 5 m M Ni 2+, both microvessel and brain preparations bound AVP with high affinity and the specific binding was saturable (Fig. 1). Scatchard analysis revealed a Ka of 2.2 ___0.4 nM and a Bmax of 49 _ 5 fmol/mg protein (means ___ S.E.M.) in pig cerebral microvessels. Total particulate fractions of the pig cerebral cortex had a Kd of 1.6+0.6 nM and a Bmax of 78-F 19 fmol/mg protein. In each of 3 sets of parallel experiments, the cortex

TABLE I SPECIFIC pH]AVP BINDING TO CEREBRAL MICROVESSELS,CORTEX AND HIPPOCAMPUS Values denote means + S.E.M. of 3 separate preparations for total particulate fractions of cerebral microvessels and cerebral cortex. Values for P2 and P3 fractions of cerebral cortex and hippocampus were obtained from a single preparation. Tissue

Ka (nM)

Bmax (frnol/mg protein)

Cerebral microvessels Total particulate fraction

2.2 __+0.4

49 __+5

Cerebral cortex Total particulate fraction P2 fraction P3 fraction

1.6 + 0.6 1.4 1.3

78 + 19 113 93

Hippocampus P2 fraction P3 fraction

1.6 0.9

93 85

124

exhibited ~ 30% higher Bmax than microvessels (Table I). Partially purified pig cortical and hippocampai membranes also exhibited high affinity specific AVP binding. Both the crude synaptosomal P2 pellets, and plasma membrane and microsomal P3 pellets from both cerebral cortex and hippocampus had similar Kd and Bmax of binding (Table I). Because of the low yield of isolated cerebral microvessels, complete structure-function studies were not practical. Therefore, we selected a few AVP analogs (at a single dose of 50 nM) to determine their ability to displace specific [3H]AVP binding. The order of potency of 50 nM of the analogs in displacing 0.7 nM [3H]AVP was: AVP, 88% displacement > (fl-mercapto-fl,fl-cyclopentamethylene propionic acid), 2(0methyl)tyrosine AVP (a potent pressor antagonist), 50% displacement > deamino AVP, 37% displacement > oxytocin, 28% displacement (Table II). In an in vivo pressor assay system which correlates with V~ receptor activity, the effectiveness rank order was: AVP > deamino AVP > oxytocin [22]. The pressor antagonist completely displaces [3H]AVP from Vi receptors with a potency equivalent to that of AVP itself. On the other hand, the antidiuretic activity of these analogs, which correlates with V2 receptor binding is: deamino AVP > AVP > > the pressor antagonist = oxytocin. Thus, the rank order of the analogs for displacing 0.7 nM [3H]AVP binding correlates well with Vt receptor characteristics rather than with those of the V~ receptor type. As in the rat brain [3, 19], our results indicate the presence of a single class of high affinity specific binding sites for AVP in pig brain microvessels, cerebral cortex and hippocampus (Table I and Fig. 1). AVP binding required the presence of a cation and, like rat brain preparations, the best cation was Ni2+; little or no binding occurred in the presence of Mn 2+ or Mg 2+. In contrast, AVP binding to rat aorta, a smooth muscle target tissue, is most effectively potentiated by Mg 2+ [18]. This strongly hints that the structure of AVP receptors, or the nature of the components that couple events subsequent to AVP receptor interaction, differ in various tissues. TABLE II E F F E C T OF AVP A N D ITS A N A L O G S ON T H E SPECIFIC B I N D I N G OF [3H]AVP TO CEREBRAL MICROVESSELS Total particulate fraction of cerebral microvessels (200/~g protein in 200/d) were incubated with 0.7 nM [3H]AVP in the absence and presence of unlabeled AVP or the indicated analogs at a concentration of 50 nM. The resulting decrease in specific [3H]AVP binding is expressed as % of control binding remaining. The AVP antagonist is (fl-mercapto-fl,fl-cyclopentamethylene propionic acid), 2(0-methyl) tyrosine AVP. Substance

Concentration (nM)

AVP bound (%)

Control AVP AVP antagonist Deamino AVP Oxytocin

0 50 50 50 50

100 12 50 63 72

125

Brain receptors in synaptosomes and microvessels are more closely related to each other than to a smooth muscle target (e.g. aorta) or to hepatocytes [ 141. The metal ion potentiation properties of AVP binding and the competition by analogs, both suggest that cerebral microvessel AVP receptors are of the Vi type. This conclusion is consistent with prior studies showing lack of increased cyclic AMP generation in isolated brain microvessels treated with AVP [ 11, 131,although adenylate cyclase in these microvessels was responsive to other hormones such as norepinephrine [l 11. Also, our postulate that brain microvessel AVP receptors are of the Vi type is supported by the demonstration of AVP-induced Ca2+ reuptake in brain capillary endothelium [12]. Brain microvessels possess diacylglycerol lipase and kinase enzyme activities which must be available if diacylglycerols are to serve as second messengers for signal transduction from Vi receptors [lo]. There is another recent report on AVP receptors in brain microvessels. Kretzschmar et al. found specific and saturable [i2%]AVP binding to microvessels of the hippocampus, but not to those of the striatum or cerebral cortex [16]. The & was 3.2 nM, which is similar to our results, but the B,,,,, of 205 fmol/mg protein is about 4-fold higher than reported here. We do not know the reason for this discrepancy but it should be noted that they used [‘251]AVP as ligand. 1251,which we presume to be on the tyrosine residue which is part of the active site, may have altered the binding properties of AVP. Only with the recent availability of high biological activity [3H]AVP has it been possible to study the binding properties of the Vi receptor [14]. Although some regional heterogeneity among brain microvessels may exist, it is unlikely that AVP receptors are restricted to microvessels of any specific brain region, because the effects of AVP on brain capillary permeability and blood flow are diffuse [2,7,21]. The existence of high affinity specific binding sites for [3H]AVP is a prerequisite for a putative role for AVP in the control of the brain microcirculation, especially BBB permeability. Our results do not allow speculation on the cellular or subcellular localization of AVP receptors. Further studies are required to delineate the ultrastructural localization and the exact functions of AVP in brain capillaries, and the mechanisms by which the effects on AVP receptors are mediated. We thank Mary Jo Mitchell for technical assistance and Jeanette Barnhart for manuscript preparation. Supported by USPHS Grant HL-35617 to S.I.H. and an American Heart Association grant to A.F.P. I Bradford, M.M., A rapid and sensitive method for the quantitation of microgram utilizing the principle of protein-dye binding, Anal. Biochem., 72 (1976) 248-254. 2 Brust, P., Changes in regional Neurochem., 46 (1986) 534541. 3 Costantini,

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