Benextramine irreversibly inhibits [125I]neuropeptide Y affinity labeling of the Y2 binding protein in bovine hippocampus

European Journal of Pharmacology - Molecular Pharmacology Sectton, 207 (1991) 89-91

© 1991 ElsevierSoence Publishers B.V. 0922-4106/91/$03.50 ADONIS 092241069100112B

89

EJPMOL 80047 Short communication

Benextramine irreversibly inhibits ! ~2sIlne~opeptide Y affinity labeling of the Y2binding protein in bovine hippocampus W. Li 1, R . G . M a c D o n a l d 2 a n d T.D. H e x u m 1 Departments of J Pharmacology and 2 Biochemtstry, Unioerstty of Nebraska Medical Center, Omaha, NE 68198-6260, U.S.A.

Received6 March 1991, accepted 12 March 1991

Affinity labeling of iodinated neuropeptide Y (NPY) to bovine hippocampal binding proteins revealed that benextramine inhibited specific NPY labeling of the 50 kDa NPY binding protein (Y2 binding protein) in a dose-dependent manner (IC50 = 33 t~M). Hippocampal membranes, which were pretreated with benextramine and washed, exhibited decreased [125I]NPY labeling of binding proteins in a similar dose-dependent manner. These findings demonstrate that benextramine irreversibly blocks specific NPY binding to the 50 kDa NPY Y2 binding protein. Neuropeptide Y (NPY); Benextramine; Y2 binding protein; Affinity labeling; Hippocampus (bovine)

1. Introduction

2. Materials and methods

Neuropeptide Y (NPY), a 36 amino acid polypeptide, is involved in regulation of both central and peripheral nervous system functions (Potter, 1988). Specific NPY binding sites have been demonstrated in brain and peripheral tissues from various species (Cherdchu et al., 1989; Sheikh et al., 1989) and subclassified as Yt and Y2 binding proteins (Wahlestedt et al., 1986). A Y2 binding protein in porcine hippocampus has been characterized through binding studies (Sheikh et al., 1989). Moreover, affinity cross-linking studies have demonstrated a specifically labeled 50 kDa NPY binding protein in porcine hippocampus (Inui et al., 1989). Binding and affinity labeling studies with bovine hippocampal membranes demonstrate the presence of a 50 kDa Y2 binding protein subtype in this tissue (Li et al., in prep.). Characteriztion of NPY binding protein function would be furthered by the development of a specific antagonist. Benextramine, a tetramine disulfide known as a non-selective, irreversible antagonist of aadrenoceptors (Benfey, 1982), has been reported to inhibit the NPY-induced increase in diastolic pressure in pithed rats and NPY binding to rat brain membranes (Doughty et al., 1990). In the present study, we provide evidence that benextramine irreversibly inhibits specific NPY affinity labeling of the 50 kDa bovine hippocampal Y2 binding protein.

2.1. M a t e r i a l s

Correspondence to: Terry D. Hexum, Ph.D., Department of Pharmacology, University of Nebraska Medical Center, 600 South 42nd Street, Omaha, NE 68198-6260, U.S.A.

Synthetic NPY was obtained from Bachem Inc., Torrance, CA, U.S.A. Mono-iodinated NPY was prepared using the chloramine T method and purified by high performance liquid chromatography (HPLC) with a specific activity of 1080 Ci/mmol. Benextramine was from Aldrich Chemical Co., Milwaukee, WI. Disuccinimidyl suberate (DSS) was from Pierce, Rockford, IL. Other reagents were from Sigma Chemical Co., St. Louis, MO. 2.2. E x p e r i m e n t a l p r o c e d u r e

Bovine hippocampus (freshly obtained from a local slaughterhouse) was minced and homogenized (Teflon pestle in glass) in 0.3 M sucrose solution containing 10 mM HEPES (pH 7.4) and 0.1 mM phenylmethylsulfonyl fluoride (PMSF). The homogenate was centrifuged at 6000 × g for 20 rain; the resultant supernatant was centrifuged at 100,000 × g for 60 rain. The pellet was washed twice with binding buffer containing 25 mM HEPES (pH 7.4), 1 mM MgCI 2, 1 mM CaC12, 1 mM PMSF and 1 m g / m l bacitracin. Membrane protein (200 ~g) was incubated with 250 pM [125I]NPY in the presence or absence of benextramine a n d / o r unlabeled NPY in the binding buffer with additional protease inhibitors (10 /~g/ml leupeptin, 10 /~g/ml antipain, 80 /~g/ml benzamidine, 20 /~g/ml aprotinin and 10 /~g/ml pepstatin A) for 90 rain at 25 o C. The membranes were centrifuged, washed and resuspended in the binding

90 buffer. [~25I]NPY degradation during the incubation was determined by HPLC to be less than 12%. DSS (1 mM) was added to initiate the cross-linking reaction. The membranes were incubated at 4 ° C for 30 min. The cross-linking reaction was stopped by the addition of 0.5 ml of 0.1 M Tris-HCl (pH 7.4). The membranes were pelleted and analyzed by SDS-polyacrylamide gel electrophoresis followed by autoradiography as described previously (Laemmli, 1970).

200

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3. Results

1

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Benextramine inhibited specific [125I]NPY labeling of the bovine hippocampal 50 kDa Y2 binding protein (fig.

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Fig. 2. The effect of benextraminepretreatment on [1251]NPYlabehng of the Y2 binding protein. Membranes (200 #g protein) were pre-incubated with different concentrations of benextrammeas indicated at 25 ° C for 40 min. Membraneswere washed three times and mcubated with 250 pM [125I]NPYin the absence or presence of 1 #M unlabeled NPY (see Methods). This autoradiogram is representaUve of three separate experiments.

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1A) in a dose-dependent m a n n e r with an IC50 of 33/~M (fig. 1B). In addition, a diffuse b a n d of protein(s) of approximately 100 kDa also appears to contain a site displaceable by unlabeled N P Y or benextramine. Benextramine was ineffective in inhibiting [125I]NPY labeling of a 58 kDa or 62 kDa protein. These and other proteins are considered non-specific b i n d i n g proteins because [125I]NPY labeling of these proteins was not inhibited by the presence of 1 # M unlabeled N P Y (fig.

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Fig. 1. (A) Benextramineinhibltmn of [125I]NPY labeling of bovine hippocampal Y2 binding protein. Membranes (200 ttg protein) were incubated with 250 pM [I251]NPY for 90 rain at 25°C m the absence or presence of the lndmatedconcentrationsof benextramineand/or 1 ~M unlabeledNPY. The samples were subjected to cross-linkingwith DSS followed by SDS-PAGE This autoradlogram is representatwe of three separate experiments. (B) The 50 kDa bands were cut from the dried gel using the autoradmgram as a template and the radioactivity determined.

The reversibility of benextramine action was investigated by treating the membranes with benextramine before NPY b i n d i n g (fig. 2). Membranes were pre-incubated in the presence or absence of different concentrations of benextramine in b i n d i n g buffer (additional protease inhibitors were present) at 2 5 ° C for 40 rain. Membranes were washed three times with binding buffer, incubated with [125I]NPY and cross-linked. [125I]NPY labeling of b i n d i n g proteins in benextraminepretreated membranes was reduced in a dose-dependent m a n n e r (fig. 2) similar to that observed in the absence of benextramine pretreatment demonstrating the irreversibility of the agent. However, addition of glutathione to washed benextramine pre-treated membranes resulted in complete recovery of specific [125I]NPY binding (data not shown).

91

4. Discussion NPY binding proteins have recently been characterized as Yt and Y2 subtypes (Wahlestedt et al., 1986). The porcine hippocampal NPY binding protein was classified as a Y2 subtype (Sheikh et al., 1989). Affinity cross-linking studies with [t2SI]NPY demonstrated the presence of a 50 kDa protein in both rat and porcine hippocampus (Inui et al., 1989; Sheikh and Williams, 1990). Binding and affinity labeling studies with bovine hippocampal membranes demonstrate that this tissue also contains a 50 kDa Y2 binding protein subtype (Li et al., in prep.). Benextramine inhibited specific [125I]NPY labeling of the bovine hippocampal 50 kDa Y2 binding protein. Moreover a diffuse band of 100 kDa protein is reduced by benextramine (as well as unlabeled NPY). However, we feel that the 50 kDa band is the principal NPY binding species based on our work (Li et al., in prep.) as well as the work of others (Sheikh et al., 1989). The specifically labeled protein bands around 100 kDa (figs. 1A and 2) could be a covalently cross-linked dimer of the 50 kDa protein or a complex of the 50 kDa protein with another unknown protein. The possible degradation of a higher molecular weight protein to the 50 kDa binding protein during membrane preparation cannot be completely eliminated. However, degradation occurring during the binding was minimized by the presence of several peptidase inhibitors and a time course experiment which indicated that no difference was detected in the density of 50 and 100 kDa bands between 1 and 3 h incubation (data not shown). The relationship of the 50 and 100 kDa proteins is currently under investigation. Benextramine is an irreversible, but non-selective, a-adrenoceptor antagonist (Benfey, 1982). Doughty et al. (1990) reported that benextramine inhibited the NPY-induced increase in diastolic pressure in pithed rats and NPY binding to rat brain membranes. Based on this report we chose to examine the effect of benextramine on the bovine hippocampal Y2 binding protein and characterize its site of action using affinity cross-linking. Our data indicate that benextramine almost completely inhibited [125I]NPY labeling of the bovine hippocampal Y2 binding protein in a dose-dependent manner with an IC50 of 33 #M. Doughty et al. (1990) reported that benextramine maximally inhibited 617o of [3H]NPY specific binding in the rat brain membranes with an IC50 of 55/~M. The observed differences in percentage inhibition may have been caused by: (a) different radioactive isotopes used ([t25I] vs. [3H]) and

the positioning of the label on the NPY molecule, (b) differences in membrane preparation, (c) species differences, or (d) differences in binding proteins present in whole brain (rat) versus a specific brain region (bovine hippocampus). Bovine hippocampal membranes pretreated with benextramine and washed exhibit a specific and irreversible reduction in [t25I]NPY labeling of the 50 kDa Y2 binding protein. Sulfhydryl groups may exist on the Y2 binding protein since benextramine is a tetramine disulfide which may interact with proteins through a disulfide-thiol interchange (Melchiorre, 1981). Benextramine-pretreated Y2 binding protein inhibition was reversed by reduced glutathione if added to benextramine-pretreated and washed membranes. These findings demonstrate that benextramine irreversibly blocks specific NPY binding to the 50 kDa NPY Y2 binding protein subtype.

Acknowledgement This work was supported by funds provided by the American Heart Association, Nebraska Affiliate.

References Benfey, B.G., 1982, Two long acting ~t-adrenoceptor blocking drugs: benextramine and phenoxybenzamine, Trends Pharmacol. Sci. 3, 470. Cherdchu, C., J.D. Deupree and T.D. Hexum, 1989, Bincling s~tes for 125I-neuropeptide Y (NPY) on membranes from bovine adrenal medulla, European J. Pharmacol. 173, 115. Doughty, M.B., S.S. Chu, D.W. Miller, K. Li and R.E. Tessel, 1990, Benextramine: a long-lasting neuropeptide Y receptor antagonist, European J. Pharmacol. 185, 113. Into, A., M. Okata, T. Inoue, N. Sakatani, M. Oya, H. Morioka, K. Shii, K. Yokono, N. Mizuno and S. Baba, 1989, Characterization of peptide YY receptors m the brain, Endocrinology 124, 402. Laemmli, U.K., 1970, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227, 680. Melchiorre, C., 1981, Tetramine disulfides: a new tool in a-adrenergic pharmacology, Trends Pharmacol. Sci. 2, 209. Potter, E.K,, 1988, Neuropeptide Y as an autonomic neurotransmitter, Pharmacol. Ther. 37, 251. Sheikh, S.P. and J.A. Williams, 1990, Structural characterization of Y1 and Y2 receptors for neuropeptide Y and peptide YY by affimty cross-linking, J. Biol. Chem. 265, 8304. Sheikh, S.P., R. H~kanson and T.W. Schwartz, 1989, Y1 and Y2 receptors for neuropeptide Y, FEBS Lett. 245, 209. Wahlestedt, C., N. Yanaihara and R. H~kanson, 1986, Evidence for different pre- and post-junctional receptors for neuropeptide Y and related peptides, Regul. Pept. 13, 307.