[3H]Inositol 1,4,5-trisphosphate binding in human cerebral cortex

~etno~ tern e Lettet~ 87 (1988) 283 287 klscx ~er Sc~cnt~tic Publishers Ireland Ltd

283

NSL 05280

[3H]Inositol 1,4,5-trisphosphate binding in human cerebral cortex L T r e v o r Y o u n g t 3 Peter P LI 1, S t e p h e n J Klsh I 2 A n d r e w S Chlu 1 and Jerry J Warsh I 3 Depattnlent~ o/~Psl~lttatt~ Pharma~ologl and 'htgtttute o/Meth~al 5~u'mes Unti~rstti o / T m o n t o C/atl, e Institute o / P ~ h t a t r ~ Toronto Ont (Canada)

IRecewed 29 No~ember 1987 Revised version received 15 January [988, Accepted 19 January 198h) Ae~ ~md~

Inosltol 1 4 5-tnsphosphate Receptor H u m a n cerebral cortex Llgand binding Brain

Specific saturable and reversible binding of [~H]lnosltol 1 4 5-tNsphosphate (IP0 was demonstrated to membranes prepared lrom autopsled cerebral cortices from 6 subjects who were free lrom psychiatric ol neurologJcal disease The binding has an affimty of 27_+8 nM ( m e a n + S E M ), capaclt,¢ (Bin,,) ol 1 09+11 18 pmol,mg protein and Js reversible m the presence of an excess ol unlabelled lP, These [~H]IP, binding slles are hkely to be physmloglcally significant receptors which merit further character~/ahon m lhe normal and diseased h u m a n brain

Phosphatldyhnosltlde-generated second messengers play an important role in neuronal transmembrane signal transductlon [4] Hydrolysis of phosphatldyhnosltol 4,5-blsphosphate by phosphohpase C leads to inosltol-l,4,5-trlsphosphate (IP0 lormarion which mobilizes calcium from the endoplasmlc retlculum (ER) in permeablhzed cells and from rat brain mlcrosomes [10, 11] Although the mechanism by which IP~ produces calcium moblhzatlon is not clear, this process may be mediated by lP~ binding to a specific membrane receptor [12, 14] Radlolabelled lP~ binding occurs in mlcrosomes from hver, pltmtary and adrenal cortex [1, 12, 13], suggesting IP~ interacts with a putative receptor on the ER membrane In addmon, specific binding of [~H]IP3 occurs in membrane preparations from several regions of rat brain [14, 15] This suggests IP~ also functions at the nerve cell membrane The potential significance of this second messenger m neuronal regulation necessitates examination and characterization of IP~ binding in human brain We report here the first demonstration of specific, saturable binding sites for IP~ m a crude membrane preparation from human postmortem frontal cortices [~H]IP~ (spec act 3 3-4 0 C1/mmol) was purchased from New England Nuclear (ol~¢ V~Omk'm e J J Warsh Section of Biochemical Psychiatry Clarke Institute of Psychhttrv 2S1)( ollegc Street Toronto, Ont Canada M 5 T 1R8

0304-3940 8h S ()q 50 @ 1988 Else,~ler Scientific Publishers Ireland Lid

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(Boston, MA) Inosltol-l-monophosphate (IP0, lnOSltol-l,4-blsphosphate (IP2) were obtained from Sigma (St Louis, MO), IP~ was purchased from Calbiochem (La Jolla, CA), and inosltol 1,3,4,5-tetraklsphosphate (IP4) was a gift from Dr R F Irvme (Cambridge, U K ) All other chemicals were of analytical pure grade Autopsled brains were obtained from 6 human subjects who were free from neurologic or psychiatric disorder The interval between death and freezing of the samples ( - 8WC) was less than 12 h in all cases Portions of frontal cortex were homogenized (Polytron setting 9, 10 s) m l0 vols of 50 mM Trls-HCI buffer (pH 8 0) containing I mM E D T A The homogenates were centrifuged at 35,000 g for l0 rain at 4 C The pellets were resuspended with the same buffer and binding assays performed immediately Binding assays contained membrane preparation (0 3-0 4 mg protein), [3H]IP3 (2 5 nM or 5 0 nM, final concn ) in 1 ml of 50 mM Trls-HCI buffer (pH 8 0) containing 1 mM E D T A and 1 mg/ml bovine serum albumin Duplicate samples were incubated at 4~C for 10 mln followed by rapid filtration through Whatman G F / B glass filters After three 3 ml washes with cold buffer, the radioactivity remaining on the filters was extracted into 7 ml Aquasol (New England Nuclear) by vigorous shaking and quantified in a Beckman liquid scintillation spectrometer (model no LS 5801) with a counting efficiency of 45-48% Non-specific binding was defined as the amount of radioactivity bound In the presence of 1 or 2 a M unlabelled IP3 when the assay was performed using 2 5 and 5 0 nM [3H]IP3, respectively Protein was measured by the method of Lowry et al [8] with bovine serum albumin as the standard The stability of [3H]IP~ under assay condmons was assessed by HPLC with a Whatman SAX-10 column as prewously described [5] and found to be greater than 90% Analyses of binding data and estimates of kinetic parameters were performed using non-linear reiterative techniques with the L I G A N D software package [9] In a typical experiment with 5 nM [3H]IP~, 1440 cpm/mg protein were bound to the membrane preparation in the absence of unlabelled IP3, whereas 180 cpm/mg protein were observed when 2/~M IP3 was added to the incubation Thus, approximately 87 5% of labelled hgand was specifically bound to the IP3 receptor in this preparatlon The binding of [3H]IP3 (2 5 nM) to human frontal cortical membrane was not altered by a 20 mm pretreatment w~th 0 5% Triton X-100 at 4'~C (data not shown) However, cortical membranes pretreated by heating (100°C, 10 ram) displayed no specific binding Specific [3H]IP3 binding was linear with respect to tissue concentrations up to 400 ag protein and maximal [3H]IP3 binding occurred between pH 8 0 and 8 5 For routine assays, binding was performed m a pH 8 0 buffer The kinetics of [3H]IP3 (2 5 aM) binding to human cortical membranes from 3 subjects were examined and yielded similar results The assocmtlon of [3H]IP~ was very rapid with half-maximal binding found by 1 mln Equlhbrium was reached by 5 rain at 4 C and remained constant for at least 30 mln Binding was reversible as evidenced by the rapid displacement of bound [~H]IP3 in the presence of an excess of unlabelted IP~ (1 a M ) For subject no 2, the rate constant for association (Kon) calculated was 2 2 8 × 105 M -I s -I, whereas that for dissociation (Koff) was 1 78x 10 2 s i From these estimates the dissociation constant (Ka =Koec/Kon) was calculated to be 71 nM

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which was comparable to the value derived from Scatchard analysis for this subject (Table I) The potency of various mosttol polyphosphates in dlsplacmg specifically bound [~H]IP~ from human cortical membranes ts shown m Fig 1 Among various mOSltOl polyphosphates tested, IP~ was the most potent with an ICs0 value of about 10 nM In comparison. IP2 and IP4 were much less potent In displacing [~H]IP~ binding (IC>0 ~ 10/lM), whereas IPi showed only marginal displacement even at high concentrations ( 100/tM) Due to limited availability of the hgand, Scatchard analysts was performed on data obtained from [3H]IP~ competition binding curves with increasing amounts of unlabelled IP~ (Inset, Fag 1), bearing m mind the specific hmitatlons of thas techmque of binding data analysis In human frontal cortex, [~H]IP3 was found to bind to a single class of receptor with a Kd of 27 _+8 0 nM (mean +_S E M ) and binding capacity (Bm~O of 1 06-1-0 18 pmol/mg protein Estimates of binding affinity and capacity for individual subjects are summarized m Table I In the present study, we have demonstrated specific, saturable and reversible bandmg of [~H]IP~ in a membrane preparation derived from human postmortem frontal cortex Thas suggests that putative IP~ receptorb occur in human neuronal membranes which can be quantified in the postmortem state The estimated banding affinity and capaclt) (Table I) are similar to those reported both in rat brain membranes [14 15] and in several peripheral tissues [1, 12, 13] However, these values can only be regarded as estimates as there is currently no mformatlon on postmortem stablhty of this putative receptor Moreover, our data are collected from a small number of subjects with widely varied ages While binding data for most of the samples analyzed were best described b!~ a sangle site model using weighted non-linear reiterative curve fitting [9], in some assays a two site model gave a significantly better fit to the data However, the valldat) of

TABLF I B I N D I N G D A T A A N D D E M O G R A P H I C S F O R I N D I V I D U A L SUBJECTS Subject

[~H]IP~

binding

Ad(nM)

Bm~,(pmol,

Age/sex (years)

Cause of death (Interval to autopsy

mg protem)

1

4 5 6

25 60 O5 17 gl 17

l l8 1 84 0 74 0 77 l 09 0 64

17 38 46 71 73 70

Mean+S E M

27_+80

109_+018

5"~-+ 10

2

M M M M M M

Acute chest trauma (6 h) Myocardial infarction {12 h) Myocardial infarction (4 h) Myocardial infarction (10 h) B r o n c h o p n e u m o m a (8 h) Superior mesenterlc artery thrombosis (8 h)

286

1 O0

cT~ cIP3

c

r5

l.,. IP2~

~A IP

(,.)

'-,l.m o

50 o o2

o._ bO

#t

o

~001.

\.

0'5 ~0 ) Bound (prnol/rng proteln) 0

,

,

-10

~

~

-8

~

-7.

"

-6

-7

,

,

-4

Inosltol Polyphosphate (M) Fig 1 Competition for [~H]IP~ binding (2 5 nM) with Inosltol polyphosphates for subject no 1 Percent specific binding is plotted against mosltol polyphosphate concentration ( - l o g M) These data werc obtained from a study performed m duphcate and are representatwe of experiments performed on brains from 3 subjects Inset shows Scatchard analysis of data on competition with unlabelled IP3 (1-500 nM) obtained m duplicate using tissue of subject no I

the two sites model ts htghly questionable smce the esUmated low affinity binding site was not resolved by more than two logartthmtc untts from the high affintty stte In addition, the estimated density of this low affimty site represented greater than 95% of total binding sites The dlssoclanon of [3H]IP3 binding from human cortical membranes (q/2 < 2 ram), was substantmlly slower than that observed m rat cerebellar membranes [14, 15] Rap~d filtraUon may m some instances be less rehable m estimating binding affimty and density with rapidly dlssocmtmg hgands In our hands, prehmmary evidence from parallel assays w~th rapid filtration or centnfugat~on of both human and rat brain membrane preparaUons gave slmdar Ka and Bmaxestimates (unpubhshed data) These observations concur w~th one other study which examined IP3 binding m rat brain [14] Addmonally, studies of IP3 binding m peripheral tissue have been successfully performed using rapid filtration [1, 12, 13] The binding of [3H]IP3 to human corttcal membranes is unhkely to be an enzyme binding site for which IP3 is a substrate The IP3-5'-phosphatase has a Km m the mlcromolar range [3], whereas IP3 phosphokmase would not be expected to occur in membranes as ~t is a soluble cytosohc enzyme [6] In addmon, the assay condmons employed here for hgand binding are subopUmal for the interaction of the substrate,

287

IP~, with the above enzymes [3, 6] Further evidence that this bmdlng s~te may be a receptor for [~H]IP~ ts the fact that the order of potency w~th which the various mos~tol polyphosphates tested here displaced specifically bound [~H]IP~ from membranes corresponds to thmr abthty to mobilize calcium from lntracellular stores [2] Considered together, these data support the hypothesis that the [~H]IP~ binding reported here represents a putative physiologically slgmficant lP~ receptor v~h~ch can be quantified in human postmortem cortical membranes The s~gntficance of a potentml IP~ receptor m human neuronal membranes has yet to be estabhshed as most studms have found specific IP~ binding in m~crosomal fractions where IP~ is known to act Hov~e~er ~t may act to gate membrane calcium channels as activation ol ~on channels by IP~ has been demonstrated m human T-lymphocyte membranes [7] In ,tn) case the present findings Indicate that the functional s~gmficance of this put,lt~X e receptor can be assessed &rectly in normal and &seased human brain The authors V~lsh to acknowledge Dr R F lrvme lor his generous gilt ot the 1Pa and Dr H N~zmk for his comments on the data We also thank B A Lem~eux 1\~ help m manuscript preparation L T Y is a MRC Fellow P P L ts an O M H F l-cllow SJ K l s a c a r e e r s c m n t l s t o f t h e O n t a n o M m l s t r y o f H e a l t h 1 Baukal A I Gtnllemelle G Rubln R P Spat A and ( an k J Binding sites lor mosltol trlspht~,,phate ill the bmm~. adrenal cortex, Bmchem Bloph~s Res ( o m m u n 1:{31198'~) 5:{2 st8 2 Bemdge M J and lrvme R F lnosltol trlsphosphate a novel second me,,senger m eellulai ~lgnal tlansduelJon N a l u r c ( L o n d ) t12(1984):{'~1 :{21 :{ Lrneux ( D e b a u x A Moreau C and Dumont, J E , Characterization ofl)-mlo-lnosllol 1 4 s-lNsphosphatt, p h o s p h a t a s e m r a t b r a m Bmchem Bu)ph)s Rcs ( o m m u n 13411988)~51 :{68 4 Fisher <, K and a, granoff B W Receptor actlxatmn and mosltol hpld h,,drolvsJ~ in neural tlssut.,, I Neuron.hem 48 (19871 999 1017 '~ Irxmc R [ Anggard E E Letcher ~ J and Do,~nes ( P Metabohsm ol mosm~l I 4 5-tHspho,,p h a t e m r a t p a r o t l d g l a n d s Bmchem J 229(1985) 505 511 6 Ir,,me R I Lcteher A J Heslop I P and Bemdge M J The mosltol tns,tetraklspho,,phatc palhway demonstration ol Ins I l 4 5)P~ :{-kmase actp~t) m ammal tissue, Nature ( k o n d ) :{2(/(1986) 6II 6t4 7 Kuno M and Gardner P Ion channels activated b~ lnoslto1 1 4,5-lnsphosphate m pla,mla membrane ol human Y-l'~mphoevtes Nature ( L o n d ) 326 (1987) 301 t04 8 Lo~r,, O H Rosebrough N J Farr, A L and Randall R J Protein measurement ~lth the Fohn phcnolreagcnt J Bu)l Chem 193(1951)265 275 9 Munson P [ and Rodbard D , L1GAND a versatile computerized approach for characteI~:atkm o[ hgand binding s'ystems Anal Blochem 107 (1980) 220 2:{9 l0 Burgess G M Receptors and phosphomosmdes m hver In 1 W Putne'~ led ) Phosphomosmdes and ReceptorMechamsms Llss, New York 1986 pp I l l 3t8 11 Shah 1 ( o h e n R S and Pant, H C InosJtol tnsphosphate-mdueed calcmm release m brain mJcrosomes BramRes 419(1987) 1 6 12 Spat A , Bradford P G McKmnev J S Rubm R P and Putnev J W , A saturable receptor tor a-~P-lnosltol-I 4 5-trlsphosphate in hepaloc}tes and neutrophlls Nature ( L o n d ) 319 {1986) s12 ~14 1:{ Spat A Lukacs G L Eberhardt 1 Kmsel L and Runnebaum B Bmdlngol mosltol phosphat~.s and mduet~on ol ( a + + release lrom pituitary mlcrosornal fractmns Bmchem J 244 ( 19871 49~ 0,96 14 Woile'¢ P F Baraban J M ( o l v m , J S and Snyder S H , lnosltol tnsphosphatc rcc~.ptor loeah:atlon xn brain ,,anable stolchemlstry with protein klnase ( Nature ( L o n d ) 325 (1987) 159 161 15 Worle,~ P F Baraban J M Supattapone S Wilson V S and Snvder S H Charaelen:,mon ol m o s ~ t o l t n s p h o s p h a l e r e c e p t o r b m d m g m b r a m J Bml Chem 262(1987) 121~2 121t6