Complex allosteric interaction of heparin with neurokinin-1 receptors

l:urrq~can Jc!a'w,d o ! Phu.e,a~ oh,,g~: . ,'<~,*h'~ueh~rPh,~rma~ oh*g) Secti.n, 207 ( I~,~l ) 2,',7 -2 70 19~;1 E h , v i c r Scieno: Publi.,hc~s [] ~.' t}¢)..~..ll',tfl/OJ'.~l)3,51} ,,II)ONI5 I ~224 [l~f,
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(.omp.be,>,'a|!oster|c" " interaction of heparin with neurokinin-I receptors TM

G a b r i c l c A ~ d r e a Knaus, H a n s - G i j n t h c r K n a u s and Alois Saria .\rr~'t:rot hcmt~tr~ ' R e w a r c h 1.',ut, Unit cr~i O Ih>spital o/F,'~ chtatCv, ,4.¢~020 lnmhruck, A u , ' m

R e c : i v e d g A p r i t 1991 accepted 14 May 1991

The effects ,,t :,c,..qM hcparin preparations on rat st~ia|al ncurokin;,>l rc~q-,,,r labelling were invcsti~,alcd. A lov. concentratio~ of hcg;:~ah; dose-dcpcntknli7 qilnulatcd [~>l]BoltonoHunlcr subslance P ([~:~IIBH-SP) binding |c, rat sttia:dal membranes. Thix cffccl 'aas mainly dtu' Io an increase ~:t the association velocity reflected by a decrease of fl~c cqu~lihrJdm dissociation rate conslan',. A higher bcparin concentration inhibited ~pccific [~-'SI]BH-SP labelling by a negative allo',tcric mechanism as a rcsul~ of an increase of the dissociatio, v< Ioc~ty. St.bstan ' , P; tleparin (binding studi¢~); Tachykimn: NK ~ rcccp|ors

I. lntroductio~t

2. Malerialls and methods

Substano,' !" is a neurolransmitter wifl~, exukatury effects o~ '.~,)~t~ ~:,eriphera~ and central neuron,;, which elicits ~ ,,~.ide variety of ~esponses on non-neuronal tissues tMaggio, 1988. Activation of neurokinin-1 (NK I } receptors is asso :iated with increased hydrolysis of ph~Jsphatid} linositol 4,5-bisphosphatc by ph ~spholipas~: C (Mantyh et al.. 19841 which indicates ~ dhcct caupling of NKj receptors t,,~ guanine n,acleotide-bindir, g regff'atory protein!; '-Fh= aim of this ~tudy was to iuvestigate the effects of several heparm preparations on NK~ recepior labellim~,. Whereas the effccts of heparin on the coagulation cascade or on lipid metabolism are reasonabb well defined, this polyanion is evidentiy involved it,. a h~.,ge number of physiologlcai processe' including cell p>Aifcration (Wright ct al., 1989) or p ~tentiafion of ang!ogcnesis (Taylor and Folkman, 1982). Heparin has been reported to interact with the adenylute cyclase syslum (Sai. man e: al., 1978) or to inhibit the inositol 1,4.5-triphosphate-activated calcium release (Ghosh et al., i988). Additiona! targets; of this pc t~an,.;n like the B-adrenoceptor kinasc (Benovic et ai., lO~,,q) or L-typ= calcium channel labelling and function (t':.naus et al., 1990) have been described. This paper demowm',aes that hcparin allosterically m o d - latcs the ir:,~:r,ction of [ 12~ tjBl-l-e,l" . . . . . . . . W|tll'~- NK~ receptors in a complex manner.

Naturally occurring, unfractionated high molecular w, ght heparin (average MW 13,600, range gtl0020f}00) :rod in the text referred to as hcparin, low moJecular weight hcp~rin (LMW hcparin, MW 4{~01)60{1(I), de-N-sulfated hcparin, crystalline bovine serum albumin (BSA) and phosphoramidone w,:re purchased from Sigma, MunidL F.R G. Unlabelled substance P was obtained from Pe~;nsula~ Laboratories, Belmont. CA. U.S.A. [I>l]Bu4ton-~hlntcr substance P was from Amersham, U.K.

Corb~agondence /~ Alnis Saria, i'4et*;*oc|',emistry Research Unit. UnivcrsRy Ilmpila!' of Ps3chialry, Amch~,!rass¢ 35, A-6020 hlllx, bruck, Auslria.

2.1. Binding studies with ! ~:~I/BH-Sf' a,'td rat striatal

Bindhlg sludics with [I>I]BH-SP were c,~entialb' performed as previously described (Humpu! et al., 199i)). Regulation of [J->IIBH-SP binding to NK~ receptors in the presence of increasing concentrations of several heparin preparations was detern,ined by incubation of 23-65 pM [IeSl]BH-SP (specific actMty !200-2 ! 75 C i / m m o l ) with 0.265-0.456 m g / m l rat striarm membrane protein until equilibrium was achieved (20 rain). All experiments wine performed at 2 0 - 2 4 ° C m 50 mM T r i s / H C i pH 7.4. 5 mM Mn~71,. 0.1 mM phen~Imethylsulfonyl fluoride ~PMSF), t;,0 nM phos. phoramidone supplemented with 200 # g / m l BSA in a final ass~,y volume of 0.25 ml. Dilution of tmlabeIled substance P or heparin derivatives was carried out in assay buffer containing 200 ~ug/ml BSA to prever, t non-apec !!k:adsorption of cor~lpou;~ds on tube walls or pipet e , ips. Assays were terminated by rapid dilution

2hS

response curee (fig. 1A). At low heparin concentrations (0.2-8 # g / m l ) NK ~ receptor labelling was dose dependently increased (EC50 value 3.14 _+ 1.21 /xg/ml) up to 173 _+ ~6% of control binding (n = 4). Higher heparin concentrations under investigation (10-1000 p,g/ml) inhibited specific [~2~I]BH-SP binding to rat striatal membranes tiCs0 value 19.41 +_3.2 /zg/ml). Low molecular weight heparin lacks only the stimulation of specific [uSI]BH-SP binding, whereas inhibition occurs in a similar range t i c s , value 13.1 +_3.76 /zg/ml). De-N-sulfated heparin was > 50-fold weaker tiCs0 value 289 +_ 45 /zg/ml), indicating the importance of

w i t h 3.5 m l i c e - c o l d 10 m M T r i s / H C l p H 7.4, 120 m M NaCI and filtered over Whatman GF/C filters (preincubatcd with 0.1% polyethylenimine). Protein concentralion was determined as d e s c r i b e d ( L o w r y et a!.. 1951) u s i n g B S A a s s t a n d a r d .

3. R e s u l t s

Heparin interacts with specific ['=Sl]BH-SP binding to rat striatal membranes in a complex manner which results at equilibrium in a biphasic, bell-shaped dose-

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Fig 1, Effects of different heparin preparations on [~251]BH-SP equilibrium binding and kinetics of rat striatal membranes. A: Rat striatal membrane protein (0.345 m g / m l ) wa~ incubated with 29.7 pM [~251]BH-SP in the presence of increasing concentrations of heparin to), LMW heparin (o) and de-N-sulfated hep~4in (~) until equilibrium was achieved. The following equilibrium binding parameters were obtained: (e) ECs01 2.84+_0.14 # g / m l , n u 1.63_+0.22. maximal stimulation up to 184%; 1Cs011 16.98_+2.54 p~g/ml, n[~ 3.01_+1.24; ( o ) 1C50 11.2_+4,56 ,~g/ml, n H 2.04 _+(I.43; (Q) ICs, 309 +_76 # g / m l ; n H 1.33 _+0.19. B: Saturation isotherm (Scatchard transformation) for neurokinin-I receptors. Rat striatal membrane protein ,0.309 m g / m l ) was incubated with [ 12Sl]BH-SP (11.89-500i pM) in the absence ( o ) and presence to) of 5 / ~ g / m l heparin. The experiments wvre performed in a final assay volume of 0.25 ml and repeated three times with essentially identical results. One representative experiment is shown. The following equilibrium binding constants were obtained. ( o ) KD1 = 69.3-+ 1.3 pM, Bm~xl = 7.82-+0.41 pM (corresponds to 25.03_+1.31 finel/mg protein); KD2=180.3_+3.4 pM, Bm,~2=3.51_+0.27 pM (corresponds to 11.35_+0.86 f m o l / m g protein); (o) KDI = 37.6_+ 1.4 pM. Bm~,~= 6.40_+0.21 pM (corresponds to 20.71 +0.67 f m o l / m g protein); KD2 = 147.6-+4.1 pM, Bronx2 = 3.22+ 0.41 pM (corresp¢mds to 10.41_+ 1.31 fmol/mg protein). C: Rat striatal membrane protein (0.424 m g / m l ) was incubated with 27.4 pM [~2SI]BH-SP for the indicated times. At equilibrium the specifically bound ligand was 2.66 pM ( o ) in the absence and 3.98 pM in the presence (o) of 5/.tg/ml or 0.18 pM ( 4, ) of 100 # g / m l heparin. The following association rate constants were measured: control Kot~er,,~d 0.085 -+0.08 rain-l; in the prese, uce of 5 /Jg/ml hor, arin K,,~,~ 0.1607-+0.11 min - l . In the presence of 100 p,g/ml heparin a Kobser~cd WaS not determined. The correspov, ding K~ 2 values taking into account receptor and ligand concentration are given in Results. D: Rat striatal membrane protein (0,365 mg/m~) was labelled with []2~t]BI-t-SP (33.4 pM) for 25 min at 22 °C. The specifically bound ligand at equilibrium was 1.87_+0.53 pM. At the times indicated dissociation was initiateJ by addition of 300 nM unlabelled substance P (o, K i1 0.198-+0.06 rain-I; K ill 0.0127-+0.006 rain-11, 300 n M substance P and 5 ~tg/ml heparin (e, K i I 0.154_+ 0.09 rain-U; K_ ill 0.0101 _+0.004 r a i n - i ) or 300 nM substance P and 100 ktg/ml heparin (~,, K iI 0,318-+ 0.{~7 rain 1: K (ll 0.0358_+0.0117 rain-u), The inset presents a semilogaritbmic transformation of the data in D. Data are expressed as means _+asympt. S.D.

269

tile N-sulfate groups. Summarizing these data suggest that both the sulfate moieties and the anionic character as well as a sufficient length of the heparin molecule are required for stimulation of specific [J25I]BH-SP labelling, To further investigate the mechanism of stimulation ot [125I]BH-SP binding to rat striatal membranes, displacement experiments with unlabelled substance P in the presence of 5 # g / m l heparin were perfor~;ed. Fig. !B exemplifies the effects of heparin on the equilibrium binding parameters of [~25I]BH-SP to rat striatal membranes expressed in a saturation isotherm. The main mechanism of the polyanion heparin could be attributed to a decrease of the apparent K D of the high-affinity ['25I]BH-SP binding site (control: K o = 74.31 + 12.4 pM; 5 / x g / m l heparin present: K D = 36.17 _+ 7.41 pM; n = 4) whereas the maximum density of binding sites was nearly unchanged. To further elucidate the mode of action by which heparin cxerts its allosteric modulation of [~25I]BH-SP binding, kinetic studies in the absence and presence of heparin were performed. Preincubation of the rat striatal membranes with 5 /xg/ml heparin resulted in a strong increase of the association velocity (fig. 1C). Under control conditions a k+~ of 0.0013 + 0.00014 min -~ pM -~ (n = 3) was determined, whereas the presence of heparin resulted in an increase of the association rate constant up to 0.00304 _+ 0.0013 m i n p M - l (n = 3). The presence of a high heparin concentration (100 p~g/ml) completely inhibited the time-dependent association of specific [~2SI]BH-SP binding. When the dissociation of the NKI r e c e p t o r / radioligand complex at equilibrium was initiated by addition of 300 nM unlabelled substance P the dissociation was biphasic; simultaneous addition of 5 p~g/ml heparin and 300 nM substance P (as well as preincubation with the same heparin concentration, not shown) resulted in an identical dissociation behavior (fig. 1D). Perturbation of equilibrium by addition of 300 nM substance P and 100 # g / m l heparin increased the dissociation rate constants. The stimulation of [125I]BH-SP binding at low heparin concentrations could be mainly attributed to a decrease of the apparent dissociation constant by a simultaneous increase of the association rate constant _..~,~,h . . .~,~ . . the dissociation kinetic parameters in the presence of 5 p.g/ml heparin remained unchanged. Higher heparin concentrations ( > t00 /xg/ml) induced the inhibitory effect on [I:5I]BH-SP binding by an increase of the dissociation velocity.

and may therefore provide an important tool to further assess the functional and structural parameters of NK~ receptors. At low heparin concentrations specific [~25I]BH-SP labelling is stimulated whereas at higher concentrations, binding is dose-dependently inhibited. When the e,.quil!brium binding parameters of [~25IJBH-SP binding in the presence of heparin are investigated, the shift to a state of higher affinity for substance P is mainly due to an increase of the association velGcity, whereas the dissociation rates remain unchanged. Higher heparin concentrations accelerate the dissociation velocity of equilibrium bound substance P argaing against a simple competitive mechanism. A possible explanation for these effects would be that heparin acts due to its high content of negative charges by general, non-specific membrane perturbation. This is, however, unlikely since heparin failed to alter specific labelling of several non-tachykinin receptors in oifferent regions of rat brain like al-adreno-, dopamine, serotonin or opiate receptors (Knaus et al., 1990"~. These findings point to the specificity of heparin action on NK~ receptors. Thus, heparin fulfills the criteria to define a novel binding domain in addition to the previously well-characterized receptor sites for the tachykinins. A direct interaction of heparin with the positively charged neuropeptide substance P seems very unlikely because it failed to adsorb on a heparin-Sepharose column even at very low ionic strength tnot shown). Summarizing we interpret our results as a complex conformational change of the NKt receptor once the heparin binding domain has been filled by the appropriate polyanion resulting in a bell-shaped displacement curve of specific [IESl]BH-SP labelling. To characterize the effects of the naturally occurring, heterodisperse polyanion heparin in more detail, further studies with defined heparin derivatives are needed to establish a clear structure-activity relatior.s! ip. Heparin and related compounds can be regarded as useful tools to further investigate the complex regulation of neurokinin receptors. Heparin could also serve as a prototype in the design of specific, non-peptidergic drugs that would exhibit selective effects on binding or regulation of tachykinin receptors.

Acknowledgements 4. Discussion

Our results establish heparin as a powerful modulator of [~251]BH-SP binding to strimal NK~ receptors

The authors gratefully acknowledge Prof. Hartmut Glossmann, Institute for Biochemical Pharmacology,Innsbruck, for helpful suggestions and facilities. This work was supported by a grant of the FWF (P'/573M) to A.S.

27{)

References Benovie, J.L.. W.C. Stone, M.G. Car m and R.J. kvfkowitz, i989. Inhibition of the /3-adrenergic r,,_eptor kinase by polyanions. J. Biol. Chem. 264, 6707. Ghosh. T.K., P.S. Eis, J.M. Mullaney, C.L. Ebert and D.L. Gill, 1988, Competitive, reversible, and potent antagonism of in(>sitol 1,4,5trisphosphate-activated calcium release by heparin, J Biol. Chem. 263, i i075. Humpel, C,, G.A. Knaus, B. Auer, I-i.G. Knaus, C. Hating, E. Theodorsson and A. Saria, 1990, Effects of haloperidol and clozapine on preprotachykinin-A messenger RNA, tachykinin tissue levels, release and neurokinin-1 receptors in the striatonigra! system, Synapse 6, 1. Knaus, H.G., F. Scheffauer, C. Romanin. H.G. Sehindlcr and tt. Glossmann, 1990, Heparin binds with high affinity to voltage-dependent L-type calcium channels - evidence for an agonistic action, J. Biol. Chem. 265, 11156.

Lowry, O.H., N.tt. Rosebrough, A.L. Farr and R.J. Randall, 1951, Protein measurement with the Folin reagent, J. Biol. Chem. 193, 265. Maggio. J.E., 1988, Tachykinins, Annu. Rev. Neurosci. 11, 13. Mantyh, P.W., R.D. Pinnock, C.P. Downes, M. Goedert and S.P. tlunt, 1984, Correlation between inositol phospholipid hydrolysis and substance P receptors in rat CNS, Nature 309, 795. Saloman, U., Y, Amir, R. Azulai and A, Amsterdam, 1978, Modulation of adenylate cyclase activity by sulfated glycosaminoglycans. 1. Inhibition by heparin of gonadotropin-stimulaled ovarian adeny!ate cyclase, Biochim. Biophys. Acta 544, 262, Taylor, S. and J. Folkman, 1982, Pro)amine is an inhibitor of angiogenesis, Nature 297, 307. Wright, Jr., T.C., J.J. Caste!lot, Jr., M. Petitou, J.C. Lormeau, J. Choay and MJ. Karnovsky, 1989, Structural determinants of heparin's growth inhibitory activity, J. Biol. Chem. 264, 1534.