3H-Substance P binding to guinea-pig ileum longitudinal smooth muscle membranes

Regulatory Peptides,

8 (1984) 273-281

273

Elsevier RPT00282

3H-Substance P binding to guinea-pig ileum longitudinal smooth muscle membranes Stephen P. Watson

*

and Leslie L. Iversen

**

M R C Neurochemical Pharmacology Unit, Medical Research Council Centre, Hills Road, Cambridge CB2 2QH, U.K.

(Received 21 December 1983; revised manuscript received 29 February 1984; accepted for publication 27 March 1984)

Summary Specific binding of 3H-substance P was studied in guinea-pig ileum longitudinal smooth muscle membranes. A single population of non-interacting sites with an apparent dissociation constant of 1.8 nM was observed. The relative potencies of some structural analogues of substance P, notably eledoisin and substance P (5-11), in competing for 3H-substance P binding sites, however, had little resemblance to their potencies in contracting the intact muscle or in eliciting the breakdown of inositol phospholipids in this tissue. The results are discussed in the light of other binding studies for substance P. substance P; receptors; tachykinins; 3H-substance P binding; physalaemin; guinea-pig ileum

Introduction Several groups have attempted to label substance P receptors using ligand binding techniques. The majority of these studies have been carried out on either membrane or intact cell preparations of pancreatic acinar cells [1], salivary glands [2-4] or brain tissues [5-9], and have used 3H-substance P, 125I-Bolton-Hunter substance P or 125I-physalaemin as the label, i.e., an agonist with identical or similar pharmacologiMolecular Biology Dept., Wellcome Research Laboratories, Burroughs Wellcome Co., 3030 Cornwallis Rd., Research Triangle Park, North Carolina 27709, U.S.A., Tel: 919 248 4464. ** Present address: Neuroscience Research Centre, Merck, Sharp & Dohme, Ltd., Hertford Road, Hoddesdon, Hertfordshire EN11 9BU, U.K. * Present address:

0167-0115/84/$03.00 © 1984 Elsevier Science Publishers B.V.

274 cal properties to substance P. All of these studies report a single population of binding sites, but there is little agreement on estimates of the dissociation constant, which range from 0.17 nM [6] to 12 nM [8]. Compounds that are structurally unrelated to substance P do not compete for the binding. However, although a number of these studies have observed a strong positive correlation between the relative potencies of substance P analogues in competing for this binding site with their biological potencies [2,5], the majority of these studies have found that a poor correlation exists for the potencies of certain substance P analogues, notably eledoisin and substance P fragments [1,3,4,6-9]. One possible explanation for these apparent discrepancies in the binding studies carried out on brain tissue is that the potencies observed have to be compared with potencies obtained in other tissues, frequently the guinea-pig ileum, and that these tissues may contain different receptor sub-types. Consequently, in the present study we have measured 3H-substance P binding in the guinea-pig ileum longitudinal smooth muscle, and compared the potencies of structural analogues of substance P in competing for this site with their potencies in contracting the smooth muscle or eliciting inositol phospholipid breakdown in the tissue.

Methods

Receptor binding assays Male guinea-pigs (300-500 g) were stunned by a blow to the head followed by bleeding. The whole length of the small intestine, except the duodenum, was removed into pre-gassed Krebs-bicarbonate solution, and longitudinal muscle strips were subsequently prepared by passing segments of the ileum over a glass rod and gently teasing the muscle away with cotton wool. The preparation of the membrane pellets and the binding experiments were carried out in a similar manner to those described for 3H-substance P binding to rat submandibular gland membranes [4]. The longitudinal muscle strips were homogenised in 10 vols. (w/v) of ice-cold buffer A (50 mM Tris-HCl, pH 7.4, containing 120 mM NaC1 and 5 mM KCI) with a Polytron set at position 7 for 15 s. The homogenate was incubated on ice in the presence of 300 mM KC1 and 10 mM EDTA for 30 min with intermittent mixing, and then centrifuged at 50 000 x g for 10 rain. The pellet was resuspended in 20 vols. of ice-cold buffer B (50 mM Tris-HCl, pH 7.4) by a Polytron at position 7 for 10 s and centrifuged again. The washing procedure was repeated twice more, and the final pellet resuspended in 60 vols. of buffer B. For binding experiments, the reaction was initiated by adding 400 /~1 of the freshly prepared membrane preparation to 100 /~1 of 50 mM Tris-HCl (pH 7.4) containing 0.02% bovine serum albumin, 1 ~tg chymostatin, 2/~g leupeptin, 20 ~g bacitracin, 3H-substance P and various test drugs. Samples were then left at 25 °C for 30 rain and the reaction terminated by adding 5 ml of ice-cold buffer A to each tube and its content filtered immediately through Whatman G F / F glass-fibre filters (pre-treated with 0.1% polyethyleneimine in water for more than 3 h in order to

275 reduce the non-specific adsorption of 3H-substance P to the filters). Each filter was then quickly washed once with 5 ml of ice-cold buffer A and radioactivity determined by liquid scintillation counting. Non-specific binding was defined using 1 #M physalaemin. The protein content of the membrane suspension was analysed by the method of Lowry et al. [10] using bovine serum albumin as the standard.

Analysis of data Scatchard plots and Hill plots for the binding of 3H-substance P to ileum longitudinal muscle membranes were generated using between 9 and 12 concentrations of 3H-substance P from 0.125-24 nM. Each experiment was analysed individually, and lines of best fit have been estimated using linear regression. Competition experiments were carried out against a concentration of 2 nM 3H-substance P, and data have been pooled from all experiments and analysed at the IC50 level. Indications of statistical significance were made using a Student's t-test. Stability of 3H-substance P and its analogues in the binding experiments The stability of 3H-substance P in the binding experiments was checked on 2 previously characterised thin layer chromatography (TLC) systems [11]: butan-l-ol/ acetic acid/water (12:5:3, v/v) and ethyl acetate/pyridine/acetic acid/water (5 : 5 : 3 : 1, v/v). These systems show good separation between 3H-substance P and its potential labelled metabolites [11]. The guinea-pig ileum was used as a bioassay to check the stability of competing analogues of substance P in the binding experiments, using procedures previously described [11]. Aliquots of the binding mixture at the termination of incubation were added to the guinea-pig ileum, and the response compared with the response produced by control samples. The actual concentration of analogue present could then be obtained from full dose response curves of that analogue on the tissue. The concentration of competing analogue in the binding mixture for these experiments was approximately 1/~M. Materials Chymostatin, leupeptin, bacitracin, bovine serum albumin (radioimmunoassay grade) and polyethyleneimine were purchased from Sigma, Poole. Substance P, eledoisin, substance P(5-11), physalaemin, substance P(1-9) amide and [pGluS,MePheS,Sarg]substance P(5-11) were purchased from Peninsula Laboratories, California. Substance P(5-11) was also obtained from Cambridge Research Biochemicals. 3H-Substance P (23 Ci/mmol) was synthesised and characterised as described previously [12]. Peptide concentrations were determined by amino acid analysis. Stock solutions of peptides were dissolved in water and stored in aliquots at - 2 0 o C; dilutions were made on the day of the experiment in Tris-HC1 containing 0.1% bovine serum albumin. 3H-Substance P was stored under liquid nitrogen until needed for experimentation when it was aliquoted out and stored at - 2 0 ° C for no more than 2 weeks.

276 Results

Properties of 3H-substance P binding Using the mixture of peptidase inhibitors described, no metabolism of 3H-substance P by guinea pig ileum longitudinal muscle membranes was detected after 30 min. This can be contrasted with the previously reported rapid and extensive degradation of 3H-substance P by guinea-pig ileum longitudinal muscle strips which occurred in the absence of inhibitors [11]. Interestingly, bacitracin alone has been previously reported to have no apparent effect on the potency of substance P on the guinea-pig ileum [11]. Under the conditions used, non-specific binding was linear with increasing concentrations of 3H-substance P and specific binding was directly proportional to the amount of tissue present. Specific binding represented approximately 40% of total 3H-substance P binding (2 nM), and was approximately 1% of the total radioactivity in the incubation. The specific binding of 3H-substance P reached equilibrium after 20-30 min. Specific binding of 3H-substance P was saturable with increasing concentrations of ligand, and Scatchard analysis of the saturation curve indicated a single population of binding sites with a dissociation constant (KD) of 1.85 + 0.27 nM (n = 5), and a density of 78.1 + 15.5 f m o l / m g protein. Hill analysis of the same data revealed a K D estimate of 1.88 + 0.24 nM and a slope of 0.89 + 0.05, which is not significantly different from 1.00. Thus, there was no evidence for negative or positive co-operativity in the binding of 3H-substance P to ileum longitudinal muscle membranes. A typical experiment is illustrated in Fig. 1.

B,

A.

30

1.0I

•

~" 24 O

0.6

~. 18

0.2

vv UJ

•

•

LI.,

~ -0.2 o. J

oen 6 0

-06 |

0

I

20

I

i

40

l

i

60

I

|

80

BOUND (fmol/mg protein)

I

%1

100

-1.C

3x10_10

I

3x10_9

I

3x!0-8

LOG CONCENTRATION (M)

Fig. 1. Analysis of specific 3H-substance P binding to guinea-pig ileum longitudinal muscle membranes obtained with increasing concentrations of 3H-substance P. (A) Scatchard analysis of the results of a typical experiment. Each point was determined in triplicate. Non-specific binding was determined using 1 pM physalaemin. (B) Hill analysis of the results of the same experiment.

277 Substance P (5-11)

lO0

% control binding

m

Eledoisin~ , ~

iI, )iMe-C7

o

60

20 0

-~o

Substance P (1-9)

-9

-8

-~

-7

Concentration

-~,

-~

(M)

Fig. 2. Displacement curves of 3H-substance P (2 nM) binding to guinea-pig ileum longitudinal muscle membranes by various agonists. Results are expressed as the mean of between 3 and 5 experiments performed in triplicate determinations. S.E.M. values were generally less than 15% of the mean.

Structure-activity profile of the 3H-substance P binding site D i s p l a c e m e n t c u r v e s f o r s u b s t a n c e P a n d its s t r u c t u r a l a n a l o g u e s w e r e g e n e r a t e d u s i n g a c o n c e n t r a t i o n o f 2 n M 3 H - s u b s t a n c e P. T h e c u r v e s w e r e p a r a l l e l a n d a p p e a r e d to b e m o n o p h a s i c w i t h H i l l c o e f f i c i e n t s n o t s i g n i f i c a n t l y d i f f e r e n t f r o m 1.0 (Fig. 2), a g a i n s u g g e s t i n g the p r e s e n c e o f a single p o p u l a t i o n o f n o n - i n t e r a c t i n g b i n d i n g sites. H o w e v e r , t h e r e l a t i v e p o t e n c i e s o f s o m e o f t h e s u b s t a n c e P a n a l o g u e s in c o m p e t i n g for this b i n d i n g site h a d little r e s e m b l a n c e to t h e i r p o t e n c i e s o n c o n t r a c t i o n a n d i n o s i t o l p h o s p h o l i p i d h y d r o l y s i s in g u i n e a - p i g i l e u m ( T a b l e I). T h e m o s t s u r p r i s i n g o b s e r v a t i o n was t h a t s u b s t a n c e P ( 5 - 1 1 ) a n d [ p G l u S , M e P h e 8, TABLE I Potencies of substance P analogues relative to substance P ( = 1) on various test systems in the guinea-pig ileum Analogue

Binding

Contraction

Substance P Eledoisin Physalaemin Substance P(5-11) [pGluS,MePheS,Sarg]substance P(5-11) Substance P(1-9) amide

1 0.006 0.26 < 4.4× 10 -5

1 0.9 1.6 0.3

< 4.4 × 10- 5 6.0 x 10 -4

0.04 2.0 × 10- 5

Inositoi phospholipid hydrolysis 1 0.78 0.84 0.13 0.03 < 1 × 10- 3

Relative potencies were derived from IC50 values for structural analogues of substance P tested against the binding of 2 nM 3H-substance P to guinea-pig ileum longitudinal muscle membranes (Fig. 2); data are relative to the experimentally determined ICs0 value for substance P (6.72 + 1.14 nM). In addition, the potencies of these analogues relative to substance P on contraction [13,14] and inositol phospholipid breakdown [15] in guinea-pig ileum are shown. The ECs0 values for substance P on contraction and inositol phospholipid hydrolysis were 1.4 nM [13] and 22 nM [15], respectively.

278 Sar9]substance P (DiMe-C7) failed to displace substance P from its binding site at concentrations up to 30 gM, although both analogues are active at nanomolar concentrations in eliciting contraction and inositol phospholipid hydrolysis in this tissue (Fig. 2, Table I). Similarly, eledoisin is approximately equipotent with substance P in eliciting ileum contraction and inositol phospholipid breakdown, and yet its potency relative to substance P on competing for 3H-substance P binding sites was only 0.006 (Fig. 2, Table I). The low potency of eledoisin and the inactivity of substance P(5-11) and DiMe-C7 did not appear to reflect their degradation, as aliquots of the incubation medium after 30 min for all three peptides produced similar or slightly smaller responses on the guinea-pig ileum than control samples. Further, 3H-DiMe-C7 has been reported to be stable when incubated in vitro with guinea-pig ileum longitudinal muscle slices for 30 rain [11].

Discussion

The lack of correlation between the relative potencies of substance P analogues in competing for 3H-substance P binding sites and their potencies in causing contraction or inositol phospholipid breakdown in guinea-pig ileum suggests the possibility that many of these structural analogues of substance P elicit contraction and inositol phospholipid hydrolysis in this tissue through a different receptor sub-type than that acted upon by substance P. Therefore, these results could be interpreted as support for the hypothesis of Lee et al. [8] that this tissue contains two distinct substance P receptor sub-types; this suggestion was formulated in order to explain the observations that certain structural analogues of substance P, including eledoisin and DiMe-C7, do not exhibit complete cross-desensitisation on the guinea-pig ileum with substance P, and that further, they recover from desensitisation induced by substance P at different rates. Alternatively, the binding may be to a site that is unrelated to the substance P receptors on the muscle membranes, e.g., it may be to a peptidase, or to a substance P receptor site that is located on other cell types found in this preparation, e.g., substance P has been shown to have potent excitatory actions on myenteric neurons in this tissue [17]. Another possible explanation for these apparent anomalous potencies is that they are the consequence of the receptor having undergone a conformational change, e.g., to an active or desensitised state, brought about, for example, by homogenisation of the tissue, or the non-physiological medium used, or because an agonist, 3H-substance P, was used to label the receptor (differences in the binding characteristics of agonists and antagonists has been previously reported for the opiate receptor [18]). These anomalous potencies, however, do not appear to reflect the rapid degradation of these analogues during the incubation. It is interesting to note that the present results bear some degree of resemblance to the majority of the substance P binding studies performed on brain and salivary glands (Table II). For example, the potency of eledoisin relative to substance P determined in the present study is 0.006 and this compares well with the previously reported values of 0.009 [6], 0.0006 [7], 0.03 [8], 0.008 [9] and 0.03 [4], but not that of

279 TABLE II Potency values of selected tachykinins and their analogues relative to substance P ( = 1) in competing for substance P binding sites in various tissues Reference

Ligand

[5]

3H-Sub- rat brain stance P 125I-Sub- mouse ruesstance P encephalic cells 3H-Sub- rat brain stance P 125I-Sub- rat cortex stance P 3H-Sub- rat brain stance P sections 3H-Sub- rat submanstance P dibutar gland 3H-Sub- guinea-pig stance P ileum

[6] [7] [8] [91 [4]

Present study

Tissue

Kd (nM)

Substance P

Eledoisin

Physalaemin

Substance P (4-11)

0.38

1

0.3

1.9

0.05

0.5

0.17

1

0.009

0.18

0.16

0.03

0.19

1

0.0006 0.009

0.003

0.001

1

0.03

0.75

0.04

-

0.5

1

0.008

0.16

-

0.003

1

1

0.03

1.5

0.21

-

1.8

1

0.006

0.26

-

12

Substance P (5-11)

< 4.4 × 10 - 5

K d = dissociation constant of 3H-substance P determined by saturation analysis.

0.3 [5] ( T a b l e II); yet, in n e a r l y all p h a r m a c o l o g i c a l assays, eledoisin is a p p r o x i m a t e l y e q u i p o t e n t or m o r e p o t e n t t h a n s u b s t a n c e P (see [16] for references). T h e m a j o r i t y o f these b i n d i n g studies, with the e x c e p t i o n of one [8], also r e p o r t very low d i s s o c i a t i o n c o n s t a n t s for the l a b e l l e d l i g a n d ( T a b l e II). These values are at least one o r d e r of m a g n i t u d e lower than the EC50 values of s u b s t a n c e P o n the m a j o r i t y of biological test systems (except the g u i n e a - p i g ileum where the existence o f s p a r e r e c e p t o r s has b e e n suggested [15]), a n d this therefore further e m p h a s i s e s the suggestions m a d e earlier that either the b i n d i n g m a y n o t b e to a r e c e p t o r site, or that the r e c e p t o r site has u n d e r g o n e a c o n f o r m a t i o n a l change. T h e p r e s e n t s t u d y is unique, however, in that it is the o n l y o n e to r e p o r t an a p p a r e n t inactivity of s u b s t a n c e P ( 5 - 1 1 ) in c o m p e t i n g for the 3 H - s u b s t a n c e P b i n d i n g site. In view of the similarities b e t w e e n the m a j o r i t y of the b i n d i n g studies, it is t e m p t i n g to suggest that they are all m o n i t o r i n g either the s a m e or closely related sites. However, p e r h a p s the o n l y strong evidence so far p r o d u c e d to suggest that this b i n d i n g site m a y b e a r e c e p t o r site is that recently r e p o r t e d b y M a n t y h et al. [19]. T h e y m o n i t o r e d the d e n s i t y of 3 H - s u b s t a n c e P b i n d i n g sites in various b r a i n regions b y a u t o r a d i o g r a p h y , a n d f o u n d a strong c o r r e l a t i o n with the m a g n i t u d e of the s u b s t a n c e P i n d u c e d b r e a k d o w n o f inositol p h o s p h o l i p i d s in these tissues. However, further evidence is n e e d e d to f i r m l y establish this conclusion, a n d p e r h a p s this c o u l d b e best o b t a i n e d t h r o u g h b i n d i n g studies using o t h e r ligands, p r e f e r a b l y antagonists, to label the receptor. U n f o r t u n a t e l y , however, the c u r r e n t of s u b s t a n c e P a n t a g o n i s t s a p p e a r s too w e a k to be used for this [20].

280

In conclusion, therefore, the present study has revealed that there is a poor correlation between the potencies of certain analogues of substance P in competing for 3H-substance P binding sites in guinea-pig ileum longitudinal muscle membranes with their effects on contraction and inositol phospholipid breakdown in this tissue. Whether this binding site is therefore a functional receptor site is still in doubt, but it would appear that binding studies using 3H-substance P as the ligand are of little use in predicting the potencies of certain structural analogues of substance P on pharmacological test systems.

Acknowledgements We would like to thank Bengt Sandberg for useful discussions, Michel Goedert for critically reading the manuscript, )nd Sue West and Mary Wynn for expert secretarial help. S.P.W. is an MRC scholar.

References 1 Jensen, R.T. and Gardner, J.D., Interaction of physalaemin, substance P and eledoisin with specific membrane receptors on pancreatic a¢inar cells, Proc. Natl. Acad. Sci. U.S.A., 76 (1979) 5679-5683. 2 Putney, J.W., Jr., Van de Walle and Wheeler, C.S., Binding of 12SI-physalaemin to rat parotid acinar cells, J. Physiol. (Lond.), 301 (1980) 205-212. 3 Liang, T. and Cascieri, M.A., Substance P receptor on parotid cell membranes, J. Neurosci., 1 (1981) 1133-1141. 4 Lee, C-M., Javitch, J.A. and Snyder, S.H., 3H-Substance P binding to salivary gland membranes: regulation by guanyl nueleotides and divalent cations, Mol. Pharmacol., 23 (1983) 563-569. 5 Hanley, M.R., Sandberg, B.E.B., Lee, C-M., Iversen, L.L., Brundish, D.E. and Wade, R., Specific binding of 3H-substance P to rat brain membranes, Nature, 286 (1980) 810-812. 6 Beaujouan, J.C., Torrens, Y., Herbet, A., Daguet, M-C., Glowinski, J. and Prochiantz, A., Specific binding of an immunoreactive and biologically active 125I-labeled substance P derivative to mouse mesencephalie cells in primary culture, Mol. Pharmacol., 22 (1982) 48-55. 7 Bittiger, H., Substance P receptors: Discussion. In: Substance P in the nervous system. CIBA Foundation symposium No. 91, 1982, Pitman Press, London, pp. 196-200. 8 Cascieri, M.A. and Liang, T., Characterization of the substance P receptor in rat brain cortex membranes and the inhibition of radioligand binding by guanine nucleotides, J. Biol. Chem., 258 (1983) 5158-5164. 9 Quirion, R., Schultz, C.W., Moody, T.W., Pert, C.B., Chase, T.N. and O'Donohue, T.L., Autoradiographic distribution of substance P receptors in rat central nervous system, Nature, 303 (1983) 714-716. 10 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 11 Watson, S.P., Rapid degradation of 3H-substance P in guinea-pig ileum and rat vas deferens in vitro, Br. J. Pharmacoi., 79 (1983) 543-552. 12 Allen, M.C., Brundish, D.E., Wade, R., Sandberg, B.E.B., Hanlcy, M.R. and Iversen, L.L., Tritiated Peptides 13. Synthesis and biological properties of [4-3H-PheS]substance P, J. Med. Chem., 25 (1982) 1209-1213. 13 Lee, C-M., Iversen, L.L., Hanley, M.R. and Sandberg, B.E.B., The possible existence of multiple receptors for substance P, Naunyn-Schndedeberg's Arch. Pharmacol., 318 (1982) 281-287.

281 14 Hanley, M.R., Lee, C-M., Watson, S.P., Sandberg, B.E.B. and lversen, L.L., Biochemical and pharmacological studies of substance P receptors. In: Protides of the biological fluids, colloquium 29, Peeters, H. (Ed.), Pergamon Press, Oxford, 1982, pp. 477-482. 15 Watson, S.P. and Downes, C.P., Substance P induced hydrolysis of inositol phospholipids in guinea-pig ileum and rat hypothalamus, Eur. J. Pharmacol., 93 (1983) 245-253. 16 Hanley, M.R. and Iversen, L.L., Substance P receptors. In: Neurotransmitter receptors, Part I, Series B, Vol. 9 Enna, S.J. and Yarnamura, H.I. (Eds.), Chapman & Hall, London, 1980, pp. 71-105. 17 Katayama, Y. and North, R.A., Does substance P mediate slow synaptic excitation within the myenteric plexus?, Nature, 274, 387-388. 18 Pert, C.B. and Snyder, S.H., Opiate receptors binding of agonists and antagonists affected differently by sodium, Mol. Pharmacol., 10 (1974) 868-879. 19 Mantyh, P.W., Pinnock, R., Downes, P., Sandberg, B., Goedert, M. and Hunt, S.P., The distribution and characterisation of substance P receptors in the central nervous system, Soc. Neurosci. Meet., Boston, 1983. 20 Rosell, S. and Fokers, K., Substance P antagonists: a new type of pharmacological tool, Trends Pharmacol. Sci., 3 (1982) 211-212.