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High-affinity 3H-substance P binding to longitudinal muscle membranes of the guinea pig small intestine
Life Sciences, Vol. 34, pp. 497-507 Printed in the U.S.A.
Pergamon Press
HIGH-AFFINITY 3H-SUBSTANCE P BINDING TO LONGITUDINAL MUSCLE MEMBRANES OF THE GUINEA PIG SMALL INTESTINE Stephen H. Buck I, Yves Maurin*,
Thomas F. Burks and Henry I. Yamamura
Departments of Pharmacology, Biochemistry, Psychiatry and the Arizona Research Laboratories, University of Arizona Health Sciences Center, Tucson, Arizona 85724 *Laboratoire de Neurochlmle, INSERM U 134, Nopital de la Salpetriere, 75634 Paris, Cedex 13, France (Received in final form November
17, 1983)
Summary The binding of 3H-substance P (3H-SP) to longitudinal muscle membranes of the guinea pig small intestine has been characterized. The binding of 3H-SP exhibited a high affinity (K d = 0.5nM). It was saturable (Bma x = 2 fmoles/mg tissue), reversible, and temperature-dependent. Kinetic studies and competition of 3H-SP blndlng by unlabeled SP yielded K d and K i values, respectively, which were in good agreement with the K d calculated from saturation studies. The binding of 3H-SP appeared to be dependent on the presence of divalent cations in the incubation buffer. It was displaced by SP and various analogs and fragments in the rank order of SP > SP-(2-11) = SP-(3-11) > Nle II- SP = physalaemin > SP-(4-11) > SP-(5-11) > eledoisin >> SP-(7-11). Our results indicate that 3H-SP binds in longitudinal muscle of the guinea pig small intestine to a biologically relevant receptor which in many respects resembles the SP receptor characterized in the brain and the salivary gland of the rat. The undecapaptlde, substance P (SP), is found throughout the small intestine of the guinea pig. The amount of SP determined by radioimmunoassay is reasonably uniform at all levels of the intestine and the longitudinal muscle with embedded myenterlc ganglia contains substantially more SP than does the mucosa (1,2). Immunohistochemical studies have demonstrated that SP in the guinea pig intestine is present in enteric neuronal cell bodies and in axonal fibers (1,3). Most of the mammalian intestinal SP content is of intrlnsic origin (4,5) but there are recent indications of an additional small extrinsic innervatlon by SP-containlng neurons in the rat and the guinea pig (6,7). The ED50 for SP in producing contraction of the guinea pig ileum i__nn vitro is in the nanomolar range making the peptide substantially more potent than acetylcholine, histamine, or 5-hydroxytryptamine (2,8). The guinea pig ileum is more sensitive than that of the rat to the contractile effects of SP and contains substantially higher levels of the peptide (2,9,10). IPresent address: Section on Biochemical Pharmacology, NHLBI-NIH, Bethesda, MD 20205. Correspondence to: Henry I. Yamamura, Ph.D., Department of Pharmacology, University of Arizona Health Sciences Center, Tucson, AZ 85724. 0024-3205/84 $3.00 + .00 Copyright (c) 1984 Pergamon Press Ltd.
498
3H-SP Binding in G.P. Small Intestine
Vol. 34, No. 5, 1984
Several recent reports have described the use of 3H-SP to identify and localize high-affinity binding sites for the peptlde in the brain and salivary gland of the rat.(ll,12,13). We have now employed minor modifications of the reported methodologies to enable us to characterize hlgh-affinlty binding sites for 3H-SP in longutudinal muscle of the guinea pig small intestine. Materials and Methods Adult Hartley or Rock guinea pigs of either sex were killed by decapitation and the entire small intestine was excised and placed in ice cold saline. Longitudinal muscle was removed by gently rubbing segments of the intestine with a cotton swab. The removed muscle from the entire intestine was homogenized with a Polytron (setting #3 for 20 sec) in 20 mM trlcine (pN 7.4) containing 120 mM NaCI and 5 mM KCI at 4°C. The clump of connective tissue that accumulated on the Polytron probe was discarded. The homogenate was centrifuged in a refrigerated centrifuge at 48,000 X g for I0 min. The pellet was resuspended in 20 mM triclne (pH 7.4) containing 300 mM KCI and I0 mM EDTA and incubated at 4°C for one hour with periodic gentle mixing. This homogenate was then centrifuged as above. The resulting pellet was washed three times in 20 mM triclne (pH 7.4) at 4°C. The pellet from the final wash was resuspended in incubation buffer at 20°C to give an 8% homogenate (based on initial wet weight of tissue) (12). Binding of 3H-SP (2-prolyl-3,4-3H(N)-SP, 28.5 Ci/mmole, New England Nuclear) was carried out in 20 mM trlclne (Sigma) (pH 7.4) containing 5 mM MnCI2, 200 ug/ml bovine serum albumin (RIA Grade, Sigma), 4 ug/ml leupeptln, 4 ug/ml chymostatln, and 40 ug/ml bacltracln (12,13). A 250-ui aliquot of the tissue homogenate was incubated with 3N-SP (usually 2 nM) in the presence or absence of i uM unlabeled SP in a final volume of 0.5 ml in polypropylene culture tubes at 20°C. To terminate the assay (usually after 20 min), 3.5 ml of 20 mM trlcine buffer (pH 7.4) containing 5 mM MnCI 2 at 4°C was added to each incubation tube and the contents of each tube was rapidly filtered under vacuum (Brandel Model M-24 Cell Harvester) over Whatman GF/B filters that had been presoaked overnight in 0.1% polyethylenimlne (Sigma). The tubes and filters were rapidly rinsed two times in an identical manner. Radioactivity on the filters was determined after incubation overnight in scintillation fluid. A liquid scintillation spectrometer which automatically determined and corrected for efficiency was employed. All experiments were carried out at least two times. All peptldes and SP fragments were purchased from Peninsula Laboratories and made up as stock solutions in ethanol containing 0.1% beta-mercaptoethanol and ahe minimum amount of water necessary for solubility. 3N-SP was stored as a stock solution similarly in an evacuated container at -20°C. Under these conditions, the amount of labeled llgand bound to tissue did not change appreciably for up to one month of storage. Trlcine buffers were prepared fresh each week. Results The effect of temperature on the muscle membranes is shown in Figure IA. higher at 20oc. At 20°C equilibrium
binding of 3H-SP to longitudinal The amount of maximum binding was was reached within 20 min. The
Vol. 34, No. 5, 1984
3H-SP Binding in G.P. Small Intestine
499
association rate constant (k+l) at 20°C was 3.5 x 106 M -I sec -I . Unless otherwise indicated, all assays were routinely carried out for 20 mln at 20°C and under these conditions using 2 nM 3H-SP and a 4% final tissue concentration, total binding was approximately 2000 DPM and non-speclflc binding (in the presence of i uM unlabeled SP) was approxlmtately 350 DPM. The difference between these values was considered speclflc binding. Presoaking of the glass fiber filters in 0.1% polyethylenlmine reduced filter binding by 90%. The specific binding was linear with tissue concentration at I rum and at 2nM 3H-SP (Figure IB), was stable for at least 40 mln at 20°C, and was abolished by first boiling the tissue. The proportion of specific binding decreased when concentrations of ligand lower or higher than 2 nM was employed and when tissue concentration was less than 4% tissue. In the absence of MnCl 2 or MgCl 2 in the incubation buffer, maximum specific binding was only 25% of total binding. The addition of either Mn ++ or Mg ++ reduced both total and non-speciflc binding while increasing the proportion of specific binding to 85% of total binding. This ionic effect was optimal at 3-10 mM cation and 5 mM MnCI 2 was thus Included routlnely in the incubation buffers.
2500
, oor ::::
•
2000
1500
100o E 500
I
0
10 TIME
I
20 (MIN.)
I
I
I
I
I
0
1%
2%
3%
4%
i
30
TISSUE CONCENTRATION
Fig. I A. Effect of temperature on the binding of 3H-SP. Incubations were carried out using 3 nM 3H-SP as described in Materials and Methods. B. Tissue linearity of 3H-SP binding. Incubations were carried out for 20 min at 20°C as described in Materials and Methods.
500
3H-SP Binding in G.P. Small Intestine
Vol. 34, No. 5, 1984
~'~ 8 0 0 0 I "" (9 Z a Z
60001v
•
4000
m
o LL
2000
UJ 0.. (D
0
i 0
i 10
I 20
I 30
3 H-SP
I 40
1 50
I 60
(nM)
Fig. 2 Saturation isotherm for the binding of 3H-SP. Incubations for 20 min at 20°C as described in Materials and Methods.
were
carried
out
Figure 2 demonstrates a saturation curve for the binding of 3H-SP. Specific binding saturated at 20-30 nM ligand. A least-squares non-linear regression analysis of the data indicated that the K d of the high-affinity site was 0.5 nM and the Bma x was 2 fmoles/mg tissue. A Scatchard plot of the saturation data is shown in Figure 3. This plot was curvilinear suggesting the possibility of multiple binding sites or states. The affinity of the possible low-affinity binding site was at least two orders of magnitude lower; however, the high amount of non-specific binding at high concentrations of 3H-SP precluded characterization of this possible low-affinity site in intestinal muscle. The Hill coefficient for the saturation data was 0.88 (Figure 3, inset).
Vol. 34, No. 5, 1984
3H-SP Binding in G.P. Small Intestine
I.j
0.2
14. ~n
501
3 4 5 LOG ['H-SI=]FREE
0.1
•
0 v
0
|
I
I
I
I
1
I
40
80
120
160
200
240
BOUND (pM)
Fig. 3 Scatchard plot and in Fig. 2.
Hill coefficient
(inset)
of
the saturation
isotherm shown
The results of a dissociation study are shown in Figure 4. The tl/2 of dissociation was approximately 4 mln but the semi-log plot of the dissociation data was curvilinear and could be resolved into k_ 1 (high) of 6.0 x 10 -4 sec -I and k_ I (low) of 2.5 x 10 -3 sec -I. The K d calculated from k+l and k_ I (high) was 0.2 nM, in close agreement with the K d obtained from the saturation isotherm data.
502
3H-SP Binding in G.P. Small Intestine
~oooI
Vol. 34, No. 5, 1984
,oo~ °
ooi / ,T
°~,
,o~
4oo[ 0
,
,
~-'e-.~
I
1
I
I
I
I
0
4
8
12
16
20
TIME
(MIN)
Fig. 4 Dissociation tlme course and seml-log binding. Incubations were carried out described in Materials and Methods.
transformation (inset) at 20°C using 2 nH
of 3H-SP 3H-SP as
TABLE I COMPETITIVE INHIBITION BY VARIOUS PEPTIDES OF 3H-SUBSTANCE P BINDING IN GUINEA PIG INTESTINAL MUSCLE MEMBRANES
Compound
IC50 (nM)
SP SP-(2-11) SP-(3-11) NLEII-sp PHYSALAEMIN
SP-(4-11) SP-(5-11) ELEDOISIN SP-7-!I )
3 10 i0 30
1 0.3 0.3 0.i
30
0.1
i00 600 i000 20~000
*IC50 of SP/IC50 of compound.
Relative Affinity*
0.03 0.005 0.003 0.00015
Vol. 34, No. 5, 1984
3H-SP Binding in G.P. Small Intestine
•
~ 80
503
•
SP
•
SP-(3-11) NLE"-SP
~0
$P-(4-11)
,T
SP-(7-111
0
0.1
|
I
1
1
I
I
1
10
100
1000
10,000
100,000
PEPTIDE CONCENTRATION
(nM)
F i g . 5. Competitive inhibition of 3H-SP binding by SP-related peptldes. Incubations were carried out for 20 mln at 20°C using 2 nM 3H-SP as described in Materials and Methods.
504
3H-SP Binding in G.P. Small Intestine
2 - - ~ ~
100
80 o ,T
-
Vol. 34, No. 5, 1984
•
A'~~%.
~
~'~_
sP
" PHYSALAEMIN
"~A
" ELEDOISIN
60
20
0 0.1
1
I
l
I
J
1
10
100
1000
10 0 0 0
PEPTIDE
CONCENTRATION
(nM)
Fig. 6 Competitive inhibition described in Fig. 5.
of
3H-SP
binding
by
tachykinlns.
Conditions
as
A comparison of the ability of various fragments and analogs of SP to competitively inhibit the binding of 3H-SP in intestinal longitudinal muscle membranes is shown in Figure 5. The IC50 for unlabeled SP was observed to be 3 nM and K i calculated with this value was 0.4 nM, in close agreement with the previous two estimates of the K d for 3H-SP. Physalaemin and eledoisin were both able to inhibit the binding of 3H-SP but physalaemin was nearly two orders of magnitude more potent (Figure 6). A comparison of the IC50's and relative affinities of all these compounds is provided in Table I.
Vol. 34, No. 5, 1984
3H-SP Binding in G.P. Small Intestine
505
Discussion The present results indicate that there are binding sites for SP in longitudinal muscle membranes of the guinea pig small intestine. The binding is destroyed by boiling and it is saturable and reversible. The K d for 3H-SP binding is 0.5 nM, which is in close agreement with the affinities recently observed for SP binding in rat brain and salivary gland (11,12,13,15). There may also be a second site in intestinal muscle with a substantially lower affinity for SP. The concentration of high-affinity binding sites in guinea pig intestinal muscle is 2 fmoles/mg tissue, which is similar to the concentration observed in rat brain (11,14,15) but somewhat lower than that in rat salivary gland (12,17). As has been reported for rat brain and salivary gland membranes (12,14), the specific binding of SP in intestinal tissue was improved by the inclusion of Mn++ or Mg++ in the incubation buffer, but this was due primarily to a reduction in non-speclfic binding. In the present investigation, the potency rank order of SP-related peptides in displacing binding (Table II) differed from that observed at 4°C in rat brain (Ii) but was in close agreement with other reports of binding in rat tissues (12,14,15). In particular, our results were very similar to those obtained by Cascieri and Liang (14) who studied the binding of 125I-BoltonHunter-SP in rat brain cerebral cortex. As previously observed in other tissues, the removal of N-terminal amino acids from SP or the substitution of Nle for Met at the C-terminus of SP reduced the ability of SP to competitively inhibit llgand binding. This indicates that both terminal regions of SP are important for optimal interaction of the molecule with its receptors (18,19). The potency rank order of SP-related peptides in inhibiting ligand binding in guinea pig longitudinal muscle was only in fair agreement with the relative ability of these compounds to produce contraction of the guinea pig ileum in vitro (Table II). The reason for this discrepancy is not clear but may involve some alteration in the tissue milieu by homogenization. It is of interest, however, that the IC50 for SP in inhibiting binding of 3H-SP in our binding assay (i.e., 3 nM) is nearly identical to the EC50 for SP in producing contraction of the guinea pig ileum (i.e., 1-3 nM) (8,16,20). Thus, in contrast to some of its related peptides, the receptor interactions of SP itself in guinea pig intestinal muscle in vitro seem to be similar in both broken membranes and intact tissue. This difference between SP and some of its relatives could be due to the existence of multiple types of SP receptors or to differential kinds of interactions of these peptides with a single type of receptor in intestinal longitudinal muscle. Based on pharmacological studies in vitro, Lee et al. (16) and Growcott et al. (21) have provided evidence for the existence of physalaemin-type (SP-P) and eledoisin-type (SP-E) SP receptors. Our binding studies in guinea pig intestinal longitudinal muscle support the contention that this tissue contains a SP-P receptor (16) and that the binding characteristics of this receptor are similar to those in rat brain and salivary gland which also apparently contain a SP-P receptor (11,12,14,15). It is temptin~ to speculate that the low-affinity, high-Bma x second binding site for °H-SP that we observed may be an SP-E receptor in longitudinal muscle. If this is the case, then it would mean that the SP-P receptor (presumably on muscle cells) is present in much lower numbers than is the SP-E receptor (which may be present on acetycholine-containing enteric neurons) (16,22,23).
506
3H-SP Binding in G.P. Small Intestine
Vol. 34, No. 5, 1984
TABLE II POTENCY RANK ORDERS OF SUBSTANCE P AND RELATED PEPTIDES
PRESENT STUDY
BINDING IN RAT CORTEX 1
BINDING IN RAT BRAIN 2
BENDING IN RAT SALIVARY GLAND 3
GUINEA PIG ILEUM4
SP
SP
SP
PHYS
SP-(3-11)
SP-(2-11); SP-(3-11) PHYS
PHYS
SP
PHYS
NLEII-sp; PHYS
SP-(2-11)
SP-(2-11)
SP-(4-11)
SP; SP-(2-11)
SP-(4-11)
sP-(3-11)
sP-(3-u)
ELED
SP-(4-11); ELED
sp-(5-11)
SP-(4-11)
NLEII-sp
SP-(7-11)
SP-(5-11)
ELED
ELED
SP-(4-11)
SP-(7-11)
se-(7-11)
SP-(5-11)
CONTRACTION
OF
SP-(7-11)
ELED SP-(7-11)
iFrom 2From 3From 4From
reference reference reference reference
14. 15. 12. 16.
Acknowledgements Susan Yamamura graciously assisted with computer analyses of our data. This investigation was supported by USPHS Grants HL-30956, DA-02163, MH-30626 and MH-27257. H.I.Y. is a recipient of a USPHS Research Scientist Development Award, Type II (MH-00095) from the National Institute of Mental Health. References i. 2. 3. 4. 5. 6. 7.
M. SCHULTZBERG, T. HOKFELT, G. NILSSON, L. TERENIUS, J.H. REHFELD~ M. BROWN, R. ELDE, M. GOLDSTEIN and S. SAID, Neurosclence 5 : 6 8 9 (1980). P. HOLZER, P.C. EMSON, L.L. IVERSEN and D.F. SHARMAN, Neurosclence 6: 1433 (1981). M. COSTA, J.B. FURNESS, l.J. LLEWELLYN-SMITH and A.C. CUELLO, Neuroscience 6 : 4 1 1 (1981). M. SCHULTZBE~, C.F. DREYFUSS, M.D. GERSHON, T. HOKFELT, R.P. ELDE, G. NILSSON, S. SAID and M. GOLDSTEIN, Brain Research 155:239 (1978). R. FRANCO, M. COSTA and J.B. FURNESS, Naunyn-Schmledeberg's Arch. Pharmacol. 3 0 7 : 5 7 (1979). A.D. HOYES and P. BARBER, Neurosci. Lett. 2 5 : 1 9 (1981). J.B. FURNESS, R.E. PAPKA, N.G. DELLA, M. COSTA and R.L. ESKAY, Neurosclence 7 : 4 4 7 (1982).
Vol. 34, No. 5, 1984
8.
9. i0.
12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.
507
S. ROSELL, U. BJORKROTH, D. CHANG, I. YAMAGUCHI, Y.-P. WAN, G. RACKUR, G. FISHER and K. FOLKERS, Substance P-Nobel Symposium 37, U.S. yon Euler and B. Pernow, eds., p. 83, Raven Press, New York (1977). G.W. TREGEAR, H.D. NIALL, J.T. POTTS, S.E. LEEMAN and M.M. CHANG, Nature New Biology 2 3 2 : 8 7 (1971). S.H. BUCK, P.P. DESHMUKH, H.I. YAMAMURA and T.F. BURKS, Neuropeptldes i: 383
ii.
3H-SP Binding in G.P. Small Intestine
(1981).
M.R. HANLEY, B.E.B. SANDBERG, C.-M. LEE, L.L. IVERSEN, D.E. BRUNDISH and R. WADE, Nature 2 8 6 : 8 1 0 (1980). C.-M. LEE, J.A. JAVITCH and S.H. SNYDER, Molec. Pharmacol. 2 3 : 5 6 3 (1983). R. QUIRION, C.W. SHULTS, T.W. MOODY, C.B. PERT, T.N. CHASE and T.L. O'DONOHUE, Nature 303:714 (1983). M.A. CASCIERI and T. LIANG, J. Biol. Chem. 258:5158 (1983). A. VIGER, J.C. BEAUJOUAN, Y. TORRENS and J. GLOWINSKI, J. Neurochem. 40: 1030 (1983). C.-M. LEE, L.L. IVERSEN, M.R. HANLEY and B.E.B. SANDBERG, Naunyn-Schmledeberg's Arch. Pharmacol. 318:281 (1982). T. LIANG and M.A. cASCIERI, J. Neuroscience i: 1133 (1981). S. LEEMAN, Dublin Substance P Meeting (1983) (in press). T. MURAKOSHI, M. YANAGISAWA, C. KITADA, M. JUJIMO and M. OTSUKA, Eur. J. Pharmacol. 9 0 : 1 3 3 (1983). R.W. BURY and M.L. MASHFORD, J. Med. Chem. 1 9 : 8 5 4 (1976). J.W. GROWCOTT, A. JAMIESON, A.V. TARPEY and L.D. TOPHAM, Eur. J. Pharmacol. 8 6 : 5 9 (1983). P. HOLZER and F. LEMBECK, Neurosci. Lett. 1 7 : i 0 1 (1980). A.B. HAWCOCK, A.G. HAYES and M.B. TYERS, Eur. J. Pharmacol. 80: 135 (1982).
Pergamon Press
HIGH-AFFINITY 3H-SUBSTANCE P BINDING TO LONGITUDINAL MUSCLE MEMBRANES OF THE GUINEA PIG SMALL INTESTINE Stephen H. Buck I, Yves Maurin*,
Thomas F. Burks and Henry I. Yamamura
Departments of Pharmacology, Biochemistry, Psychiatry and the Arizona Research Laboratories, University of Arizona Health Sciences Center, Tucson, Arizona 85724 *Laboratoire de Neurochlmle, INSERM U 134, Nopital de la Salpetriere, 75634 Paris, Cedex 13, France (Received in final form November
17, 1983)
Summary The binding of 3H-substance P (3H-SP) to longitudinal muscle membranes of the guinea pig small intestine has been characterized. The binding of 3H-SP exhibited a high affinity (K d = 0.5nM). It was saturable (Bma x = 2 fmoles/mg tissue), reversible, and temperature-dependent. Kinetic studies and competition of 3H-SP blndlng by unlabeled SP yielded K d and K i values, respectively, which were in good agreement with the K d calculated from saturation studies. The binding of 3H-SP appeared to be dependent on the presence of divalent cations in the incubation buffer. It was displaced by SP and various analogs and fragments in the rank order of SP > SP-(2-11) = SP-(3-11) > Nle II- SP = physalaemin > SP-(4-11) > SP-(5-11) > eledoisin >> SP-(7-11). Our results indicate that 3H-SP binds in longitudinal muscle of the guinea pig small intestine to a biologically relevant receptor which in many respects resembles the SP receptor characterized in the brain and the salivary gland of the rat. The undecapaptlde, substance P (SP), is found throughout the small intestine of the guinea pig. The amount of SP determined by radioimmunoassay is reasonably uniform at all levels of the intestine and the longitudinal muscle with embedded myenterlc ganglia contains substantially more SP than does the mucosa (1,2). Immunohistochemical studies have demonstrated that SP in the guinea pig intestine is present in enteric neuronal cell bodies and in axonal fibers (1,3). Most of the mammalian intestinal SP content is of intrlnsic origin (4,5) but there are recent indications of an additional small extrinsic innervatlon by SP-containlng neurons in the rat and the guinea pig (6,7). The ED50 for SP in producing contraction of the guinea pig ileum i__nn vitro is in the nanomolar range making the peptide substantially more potent than acetylcholine, histamine, or 5-hydroxytryptamine (2,8). The guinea pig ileum is more sensitive than that of the rat to the contractile effects of SP and contains substantially higher levels of the peptide (2,9,10). IPresent address: Section on Biochemical Pharmacology, NHLBI-NIH, Bethesda, MD 20205. Correspondence to: Henry I. Yamamura, Ph.D., Department of Pharmacology, University of Arizona Health Sciences Center, Tucson, AZ 85724. 0024-3205/84 $3.00 + .00 Copyright (c) 1984 Pergamon Press Ltd.
498
3H-SP Binding in G.P. Small Intestine
Vol. 34, No. 5, 1984
Several recent reports have described the use of 3H-SP to identify and localize high-affinity binding sites for the peptlde in the brain and salivary gland of the rat.(ll,12,13). We have now employed minor modifications of the reported methodologies to enable us to characterize hlgh-affinlty binding sites for 3H-SP in longutudinal muscle of the guinea pig small intestine. Materials and Methods Adult Hartley or Rock guinea pigs of either sex were killed by decapitation and the entire small intestine was excised and placed in ice cold saline. Longitudinal muscle was removed by gently rubbing segments of the intestine with a cotton swab. The removed muscle from the entire intestine was homogenized with a Polytron (setting #3 for 20 sec) in 20 mM trlcine (pN 7.4) containing 120 mM NaCI and 5 mM KCI at 4°C. The clump of connective tissue that accumulated on the Polytron probe was discarded. The homogenate was centrifuged in a refrigerated centrifuge at 48,000 X g for I0 min. The pellet was resuspended in 20 mM triclne (pH 7.4) containing 300 mM KCI and I0 mM EDTA and incubated at 4°C for one hour with periodic gentle mixing. This homogenate was then centrifuged as above. The resulting pellet was washed three times in 20 mM triclne (pH 7.4) at 4°C. The pellet from the final wash was resuspended in incubation buffer at 20°C to give an 8% homogenate (based on initial wet weight of tissue) (12). Binding of 3H-SP (2-prolyl-3,4-3H(N)-SP, 28.5 Ci/mmole, New England Nuclear) was carried out in 20 mM trlclne (Sigma) (pH 7.4) containing 5 mM MnCI2, 200 ug/ml bovine serum albumin (RIA Grade, Sigma), 4 ug/ml leupeptln, 4 ug/ml chymostatln, and 40 ug/ml bacltracln (12,13). A 250-ui aliquot of the tissue homogenate was incubated with 3N-SP (usually 2 nM) in the presence or absence of i uM unlabeled SP in a final volume of 0.5 ml in polypropylene culture tubes at 20°C. To terminate the assay (usually after 20 min), 3.5 ml of 20 mM trlcine buffer (pH 7.4) containing 5 mM MnCI 2 at 4°C was added to each incubation tube and the contents of each tube was rapidly filtered under vacuum (Brandel Model M-24 Cell Harvester) over Whatman GF/B filters that had been presoaked overnight in 0.1% polyethylenimlne (Sigma). The tubes and filters were rapidly rinsed two times in an identical manner. Radioactivity on the filters was determined after incubation overnight in scintillation fluid. A liquid scintillation spectrometer which automatically determined and corrected for efficiency was employed. All experiments were carried out at least two times. All peptldes and SP fragments were purchased from Peninsula Laboratories and made up as stock solutions in ethanol containing 0.1% beta-mercaptoethanol and ahe minimum amount of water necessary for solubility. 3N-SP was stored as a stock solution similarly in an evacuated container at -20°C. Under these conditions, the amount of labeled llgand bound to tissue did not change appreciably for up to one month of storage. Trlcine buffers were prepared fresh each week. Results The effect of temperature on the muscle membranes is shown in Figure IA. higher at 20oc. At 20°C equilibrium
binding of 3H-SP to longitudinal The amount of maximum binding was was reached within 20 min. The
Vol. 34, No. 5, 1984
3H-SP Binding in G.P. Small Intestine
499
association rate constant (k+l) at 20°C was 3.5 x 106 M -I sec -I . Unless otherwise indicated, all assays were routinely carried out for 20 mln at 20°C and under these conditions using 2 nM 3H-SP and a 4% final tissue concentration, total binding was approximately 2000 DPM and non-speclflc binding (in the presence of i uM unlabeled SP) was approxlmtately 350 DPM. The difference between these values was considered speclflc binding. Presoaking of the glass fiber filters in 0.1% polyethylenlmine reduced filter binding by 90%. The specific binding was linear with tissue concentration at I rum and at 2nM 3H-SP (Figure IB), was stable for at least 40 mln at 20°C, and was abolished by first boiling the tissue. The proportion of specific binding decreased when concentrations of ligand lower or higher than 2 nM was employed and when tissue concentration was less than 4% tissue. In the absence of MnCl 2 or MgCl 2 in the incubation buffer, maximum specific binding was only 25% of total binding. The addition of either Mn ++ or Mg ++ reduced both total and non-speciflc binding while increasing the proportion of specific binding to 85% of total binding. This ionic effect was optimal at 3-10 mM cation and 5 mM MnCI 2 was thus Included routlnely in the incubation buffers.
2500
, oor ::::
•
2000
1500
100o E 500
I
0
10 TIME
I
20 (MIN.)
I
I
I
I
I
0
1%
2%
3%
4%
i
30
TISSUE CONCENTRATION
Fig. I A. Effect of temperature on the binding of 3H-SP. Incubations were carried out using 3 nM 3H-SP as described in Materials and Methods. B. Tissue linearity of 3H-SP binding. Incubations were carried out for 20 min at 20°C as described in Materials and Methods.
500
3H-SP Binding in G.P. Small Intestine
Vol. 34, No. 5, 1984
~'~ 8 0 0 0 I "" (9 Z a Z
60001v
•
4000
m
o LL
2000
UJ 0.. (D
0
i 0
i 10
I 20
I 30
3 H-SP
I 40
1 50
I 60
(nM)
Fig. 2 Saturation isotherm for the binding of 3H-SP. Incubations for 20 min at 20°C as described in Materials and Methods.
were
carried
out
Figure 2 demonstrates a saturation curve for the binding of 3H-SP. Specific binding saturated at 20-30 nM ligand. A least-squares non-linear regression analysis of the data indicated that the K d of the high-affinity site was 0.5 nM and the Bma x was 2 fmoles/mg tissue. A Scatchard plot of the saturation data is shown in Figure 3. This plot was curvilinear suggesting the possibility of multiple binding sites or states. The affinity of the possible low-affinity binding site was at least two orders of magnitude lower; however, the high amount of non-specific binding at high concentrations of 3H-SP precluded characterization of this possible low-affinity site in intestinal muscle. The Hill coefficient for the saturation data was 0.88 (Figure 3, inset).
Vol. 34, No. 5, 1984
3H-SP Binding in G.P. Small Intestine
I.j
0.2
14. ~n
501
3 4 5 LOG ['H-SI=]FREE
0.1
•
0 v
0
|
I
I
I
I
1
I
40
80
120
160
200
240
BOUND (pM)
Fig. 3 Scatchard plot and in Fig. 2.
Hill coefficient
(inset)
of
the saturation
isotherm shown
The results of a dissociation study are shown in Figure 4. The tl/2 of dissociation was approximately 4 mln but the semi-log plot of the dissociation data was curvilinear and could be resolved into k_ 1 (high) of 6.0 x 10 -4 sec -I and k_ I (low) of 2.5 x 10 -3 sec -I. The K d calculated from k+l and k_ I (high) was 0.2 nM, in close agreement with the K d obtained from the saturation isotherm data.
502
3H-SP Binding in G.P. Small Intestine
~oooI
Vol. 34, No. 5, 1984
,oo~ °
ooi / ,T
°~,
,o~
4oo[ 0
,
,
~-'e-.~
I
1
I
I
I
I
0
4
8
12
16
20
TIME
(MIN)
Fig. 4 Dissociation tlme course and seml-log binding. Incubations were carried out described in Materials and Methods.
transformation (inset) at 20°C using 2 nH
of 3H-SP 3H-SP as
TABLE I COMPETITIVE INHIBITION BY VARIOUS PEPTIDES OF 3H-SUBSTANCE P BINDING IN GUINEA PIG INTESTINAL MUSCLE MEMBRANES
Compound
IC50 (nM)
SP SP-(2-11) SP-(3-11) NLEII-sp PHYSALAEMIN
SP-(4-11) SP-(5-11) ELEDOISIN SP-7-!I )
3 10 i0 30
1 0.3 0.3 0.i
30
0.1
i00 600 i000 20~000
*IC50 of SP/IC50 of compound.
Relative Affinity*
0.03 0.005 0.003 0.00015
Vol. 34, No. 5, 1984
3H-SP Binding in G.P. Small Intestine
•
~ 80
503
•
SP
•
SP-(3-11) NLE"-SP
~0
$P-(4-11)
,T
SP-(7-111
0
0.1
|
I
1
1
I
I
1
10
100
1000
10,000
100,000
PEPTIDE CONCENTRATION
(nM)
F i g . 5. Competitive inhibition of 3H-SP binding by SP-related peptldes. Incubations were carried out for 20 mln at 20°C using 2 nM 3H-SP as described in Materials and Methods.
504
3H-SP Binding in G.P. Small Intestine
2 - - ~ ~
100
80 o ,T
-
Vol. 34, No. 5, 1984
•
A'~~%.
~
~'~_
sP
" PHYSALAEMIN
"~A
" ELEDOISIN
60
20
0 0.1
1
I
l
I
J
1
10
100
1000
10 0 0 0
PEPTIDE
CONCENTRATION
(nM)
Fig. 6 Competitive inhibition described in Fig. 5.
of
3H-SP
binding
by
tachykinlns.
Conditions
as
A comparison of the ability of various fragments and analogs of SP to competitively inhibit the binding of 3H-SP in intestinal longitudinal muscle membranes is shown in Figure 5. The IC50 for unlabeled SP was observed to be 3 nM and K i calculated with this value was 0.4 nM, in close agreement with the previous two estimates of the K d for 3H-SP. Physalaemin and eledoisin were both able to inhibit the binding of 3H-SP but physalaemin was nearly two orders of magnitude more potent (Figure 6). A comparison of the IC50's and relative affinities of all these compounds is provided in Table I.
Vol. 34, No. 5, 1984
3H-SP Binding in G.P. Small Intestine
505
Discussion The present results indicate that there are binding sites for SP in longitudinal muscle membranes of the guinea pig small intestine. The binding is destroyed by boiling and it is saturable and reversible. The K d for 3H-SP binding is 0.5 nM, which is in close agreement with the affinities recently observed for SP binding in rat brain and salivary gland (11,12,13,15). There may also be a second site in intestinal muscle with a substantially lower affinity for SP. The concentration of high-affinity binding sites in guinea pig intestinal muscle is 2 fmoles/mg tissue, which is similar to the concentration observed in rat brain (11,14,15) but somewhat lower than that in rat salivary gland (12,17). As has been reported for rat brain and salivary gland membranes (12,14), the specific binding of SP in intestinal tissue was improved by the inclusion of Mn++ or Mg++ in the incubation buffer, but this was due primarily to a reduction in non-speclfic binding. In the present investigation, the potency rank order of SP-related peptides in displacing binding (Table II) differed from that observed at 4°C in rat brain (Ii) but was in close agreement with other reports of binding in rat tissues (12,14,15). In particular, our results were very similar to those obtained by Cascieri and Liang (14) who studied the binding of 125I-BoltonHunter-SP in rat brain cerebral cortex. As previously observed in other tissues, the removal of N-terminal amino acids from SP or the substitution of Nle for Met at the C-terminus of SP reduced the ability of SP to competitively inhibit llgand binding. This indicates that both terminal regions of SP are important for optimal interaction of the molecule with its receptors (18,19). The potency rank order of SP-related peptides in inhibiting ligand binding in guinea pig longitudinal muscle was only in fair agreement with the relative ability of these compounds to produce contraction of the guinea pig ileum in vitro (Table II). The reason for this discrepancy is not clear but may involve some alteration in the tissue milieu by homogenization. It is of interest, however, that the IC50 for SP in inhibiting binding of 3H-SP in our binding assay (i.e., 3 nM) is nearly identical to the EC50 for SP in producing contraction of the guinea pig ileum (i.e., 1-3 nM) (8,16,20). Thus, in contrast to some of its related peptides, the receptor interactions of SP itself in guinea pig intestinal muscle in vitro seem to be similar in both broken membranes and intact tissue. This difference between SP and some of its relatives could be due to the existence of multiple types of SP receptors or to differential kinds of interactions of these peptides with a single type of receptor in intestinal longitudinal muscle. Based on pharmacological studies in vitro, Lee et al. (16) and Growcott et al. (21) have provided evidence for the existence of physalaemin-type (SP-P) and eledoisin-type (SP-E) SP receptors. Our binding studies in guinea pig intestinal longitudinal muscle support the contention that this tissue contains a SP-P receptor (16) and that the binding characteristics of this receptor are similar to those in rat brain and salivary gland which also apparently contain a SP-P receptor (11,12,14,15). It is temptin~ to speculate that the low-affinity, high-Bma x second binding site for °H-SP that we observed may be an SP-E receptor in longitudinal muscle. If this is the case, then it would mean that the SP-P receptor (presumably on muscle cells) is present in much lower numbers than is the SP-E receptor (which may be present on acetycholine-containing enteric neurons) (16,22,23).
506
3H-SP Binding in G.P. Small Intestine
Vol. 34, No. 5, 1984
TABLE II POTENCY RANK ORDERS OF SUBSTANCE P AND RELATED PEPTIDES
PRESENT STUDY
BINDING IN RAT CORTEX 1
BINDING IN RAT BRAIN 2
BENDING IN RAT SALIVARY GLAND 3
GUINEA PIG ILEUM4
SP
SP
SP
PHYS
SP-(3-11)
SP-(2-11); SP-(3-11) PHYS
PHYS
SP
PHYS
NLEII-sp; PHYS
SP-(2-11)
SP-(2-11)
SP-(4-11)
SP; SP-(2-11)
SP-(4-11)
sP-(3-11)
sP-(3-u)
ELED
SP-(4-11); ELED
sp-(5-11)
SP-(4-11)
NLEII-sp
SP-(7-11)
SP-(5-11)
ELED
ELED
SP-(4-11)
SP-(7-11)
se-(7-11)
SP-(5-11)
CONTRACTION
OF
SP-(7-11)
ELED SP-(7-11)
iFrom 2From 3From 4From
reference reference reference reference
14. 15. 12. 16.
Acknowledgements Susan Yamamura graciously assisted with computer analyses of our data. This investigation was supported by USPHS Grants HL-30956, DA-02163, MH-30626 and MH-27257. H.I.Y. is a recipient of a USPHS Research Scientist Development Award, Type II (MH-00095) from the National Institute of Mental Health. References i. 2. 3. 4. 5. 6. 7.
M. SCHULTZBERG, T. HOKFELT, G. NILSSON, L. TERENIUS, J.H. REHFELD~ M. BROWN, R. ELDE, M. GOLDSTEIN and S. SAID, Neurosclence 5 : 6 8 9 (1980). P. HOLZER, P.C. EMSON, L.L. IVERSEN and D.F. SHARMAN, Neurosclence 6: 1433 (1981). M. COSTA, J.B. FURNESS, l.J. LLEWELLYN-SMITH and A.C. CUELLO, Neuroscience 6 : 4 1 1 (1981). M. SCHULTZBE~, C.F. DREYFUSS, M.D. GERSHON, T. HOKFELT, R.P. ELDE, G. NILSSON, S. SAID and M. GOLDSTEIN, Brain Research 155:239 (1978). R. FRANCO, M. COSTA and J.B. FURNESS, Naunyn-Schmledeberg's Arch. Pharmacol. 3 0 7 : 5 7 (1979). A.D. HOYES and P. BARBER, Neurosci. Lett. 2 5 : 1 9 (1981). J.B. FURNESS, R.E. PAPKA, N.G. DELLA, M. COSTA and R.L. ESKAY, Neurosclence 7 : 4 4 7 (1982).
Vol. 34, No. 5, 1984
8.
9. i0.
12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.
507
S. ROSELL, U. BJORKROTH, D. CHANG, I. YAMAGUCHI, Y.-P. WAN, G. RACKUR, G. FISHER and K. FOLKERS, Substance P-Nobel Symposium 37, U.S. yon Euler and B. Pernow, eds., p. 83, Raven Press, New York (1977). G.W. TREGEAR, H.D. NIALL, J.T. POTTS, S.E. LEEMAN and M.M. CHANG, Nature New Biology 2 3 2 : 8 7 (1971). S.H. BUCK, P.P. DESHMUKH, H.I. YAMAMURA and T.F. BURKS, Neuropeptldes i: 383
ii.
3H-SP Binding in G.P. Small Intestine
(1981).
M.R. HANLEY, B.E.B. SANDBERG, C.-M. LEE, L.L. IVERSEN, D.E. BRUNDISH and R. WADE, Nature 2 8 6 : 8 1 0 (1980). C.-M. LEE, J.A. JAVITCH and S.H. SNYDER, Molec. Pharmacol. 2 3 : 5 6 3 (1983). R. QUIRION, C.W. SHULTS, T.W. MOODY, C.B. PERT, T.N. CHASE and T.L. O'DONOHUE, Nature 303:714 (1983). M.A. CASCIERI and T. LIANG, J. Biol. Chem. 258:5158 (1983). A. VIGER, J.C. BEAUJOUAN, Y. TORRENS and J. GLOWINSKI, J. Neurochem. 40: 1030 (1983). C.-M. LEE, L.L. IVERSEN, M.R. HANLEY and B.E.B. SANDBERG, Naunyn-Schmledeberg's Arch. Pharmacol. 318:281 (1982). T. LIANG and M.A. cASCIERI, J. Neuroscience i: 1133 (1981). S. LEEMAN, Dublin Substance P Meeting (1983) (in press). T. MURAKOSHI, M. YANAGISAWA, C. KITADA, M. JUJIMO and M. OTSUKA, Eur. J. Pharmacol. 9 0 : 1 3 3 (1983). R.W. BURY and M.L. MASHFORD, J. Med. Chem. 1 9 : 8 5 4 (1976). J.W. GROWCOTT, A. JAMIESON, A.V. TARPEY and L.D. TOPHAM, Eur. J. Pharmacol. 8 6 : 5 9 (1983). P. HOLZER and F. LEMBECK, Neurosci. Lett. 1 7 : i 0 1 (1980). A.B. HAWCOCK, A.G. HAYES and M.B. TYERS, Eur. J. Pharmacol. 80: 135 (1982).