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Characteristics of 3H-substance P binding sites in rat brain membranes
Peptide~. Vol. 5. pp. 833-836, 1984. :~"Ankho International Inc. Printed in the U.S.A.
0196-9781/84 $3.00 + ,00
BRIEF COMMUNICATION
Characteristics of 3H-Substance P Binding Sites in Rat Brain Membranes YOUSEF
C H A N H . P A R K , * V. J O H N M A S S A R I , * R E M I Q U I R I O N , * * TIZABI,* CLIFFORD W. SHULTS* AND THOMAS L. O'DONOHUE*
*Department o f Pharmacology, College o f Medicine, Howard University 520 W. Streeet, N.W., Washington, D.C. 20059 *Experimental Therapeutics Branch. National Institute o f Neurological and Communicative Disorders and Stroke, Bethesda, MD 20205 $Section on Brain Biochemistry. Clinical Neuroscience Branch, National Institute o f Mental Health Bethesda, MID 20205 R e c e i v e d 30 J u n e 1983 PARK, C. H., V. J. MASSARI. R. QUIRION, Y. TIZABI. C. W. SHULTS AND T. L. O'DONOHUE. Characteristics of :tH-substance P binding sites in rat brain membranes. PEFTIDES 5(4) 833--836, 1984.--Binding characteristics of SHSubstance P (SP) were studied with rat brain membranes using a method applied to peripheral tissues by Lee and Snyder [15]. This method was well applicable to central nervous system (CNS) tissues. The results in the present study indicate that specific :~H-SP binding reaches a plateau only after 20 minutes of incubation, and the binding sites are saturable at a relatively low concentration of aH-SP. Scatchard analysis of specific binding data reveals a single class of binding sites with a high affinity (Kd-0.30 nM) and a low density (Bmax--27.7 fmol/mg protein) in rat brain membranes. A Hill plot of the displacement curve of :'H-SP with unlabelled SP showed no indication for cooperativity (n,=0.83). The relative potencies of binding of various SP fragments at :'H-SP binding sites were fairly parallel to the length of the C-terminal frasments. Neurotransmitters not structurally related to SP produced no effect on ~H-SP binding even when used at mieromolar concentrations. Rat brain membranes
:'H-SP binding sites
Specific binding
O N E of the important neuropeptides found in the nervous system is the undecapeptide Substance P (SP). Currently, several lines o f evidence are available to support the hypothesis that SP plays a role as a neurotransmitter or modulator in the central nervous system (CNS). as well as in the periphery. Thus, SP has an uneven distribution, [4,19], is synthesized in neurons [ 1I, 28, 30] and can be released by drugs or electrical stimuli. SP has also been shown to exert a variety o f physiological actions such as depolarization of spinal cord motoneurons, [12,22] potent vasodilation, [16] smooth muscle contraction, [29] and salivation [17] and sensory transmission in the spinal cord [8,10]. In recent years, the apparent physiological importance of SP has led investigators to attempt to characterize SP receptors in various systems including the mammalian CNS. In light of the existence of multiple receptor subtypes for some neurotransmitters and the variety o f physiological functions of SP in different systems, it is possible that multiple subtypes o f SP receptors may exist. In fact, based mainly on the disparities in the relative potencies of SP fragments and SP related peptides in various peripheral tissues, several groups have suggested that subclasses of SP receptors exist [5, 17,
18, 29]. It is not certain, however, whether this is the case within the mammalian CNS. Two groups have attempted to characterize SP receptors in the CNS by using 3H-SP as the labelled ligand in a homogenization assay [6,21]. The interpretation o f these studies is difficult, however, due to certain methodological problems encountered in the studies. Nakata et al. [21], for example, incubated tissue at 0°C for only one minute, which would probably be an insufficient time for maximal equilibrium binding o f 3H-SP to membranes. Likewise, Hanley [6] et al. employed a '~H-SP concentration for most o f their assays in which considerably higher nonspecific than specific binding was obtained. A high ratio of nonspecific to specific binding would make it difficult to detect potential low affinity binding sites. Finally, there are substantial discrepancies on the pharmacology o f 3H-SP binding sites in the mammalian CNS, depending on whether homogenization [6,21] or slice binding methodologies were employed [25]. Therefore, in this report, we have made an attempt to further explore the properties of SP receptors in the rat brain using a method applied to salivary gland membranes by Lee and Synder [15]. This method was used because it is also
833
834
PARK ET AL.
applicable to CNS tissues, and it generates a favorable ratio of specific over nonspecific binding. In the present paper, we report there is a single class of binding sites with a high affinity and a low density in rat brain membranes. These binding sites did not show cooperativity for the binding of SP. The binding site seemed highly specific for the C-terminus of SP and had a relatively high a£fnity for physalaemin but a low affinity for eledoisin. The properties of the binding sites for sH-SP appear to reflect those expected for SP receptors. METHOD
Membrane Preparation
The binding method of Lee and Synder [15] was used with a slight modification. Whole brains (except cerebelli) were removed on ice from adult male Sprague-Dawley rats and were stored at -80"C for no longer than one week. The brain tissue was homogenized in 10 vol. of ice-cold buffer A (50 mM Tris-HCl, pH=7.40, 120 raM NaCI and 5 mM KCI) with a Tissumizer (Tekmar Co.) for 20 seconds. The homogenate was centrifuged at 50,000xg for l0 minutes at 40C and the supernatant was discarded. The pellet was resuspended in ice-cold tris buffer (50 mM Tris-HCl, pH-~7.4, 300 mM KCI and 10 mM EDTA) using a sonicator before being incubated on ice for 30 minutes with gentle shaking. After centrifugation as above, the pellet was resuspended in 20 vol. of icecold buffer B (50 mM Tris-HCl, pH=7.4), centrifuged again in the same way and the supernatant was discarded. The pellet was washed in buffer B one more time repeating the same procedure as above before f'mal resuspension in 60 vol. of buffer B. Aliquots were taken for protein determination by the method of Lowry et al. [20]. Total binding was determined by adding 200/~l of final membrane suspension into tubes containing 200 ~1 of various concentrations of 3H-SP and 200 ~l of buffer containing protease inhibitors and MnCl~ (50 mM Tris-HCi, pH=7.4, 0.02% BSA, Chymostatin 0.2 rag/100 ml, Leupeptin 0.4 rag/100 mi, Bacitracin 4 rag/100 ml and 3 mM MnCI0. MnCl~ has been shown to increase specific binding of 3H-SP to peripheral tissue [15]. For the determination of nonspecific binding, unlabeled SP (prepared in the same buffer as above) was added to a final concentration of I ~M. The incubation, which was initiated by adding the membrane suspension, was performed for 20 minutes at 20°C with gentle shaking, and terminated with 5 ml of ice-cold buffer A. The tubes contents were filtered immediately through Whatman GF/C glass fiber filters under vacuum. In order to reduce nonspecific binding the filters were presoaked in distilled water containing 0.1% polyethylenimine for several hours prior to use. After the f'dtration, each of the tubes and rflters was washed once with 5 ml of ice-cold buffer A. Each filter was placed in 7 ml of Insta-gel (Packard Co.) scintillant before being counted by liquid scintillation spectrometry. Specific binding was calculated as the difference between total and nonspecific binding. MATERIALS
:~H-SP (SP) (specific activity=28.5 Ci/m Mol) was purchased from New England Nuclear at -800C until use. Chymostatin, leupeptin, bacitracin, bovine serum albumin, polethyleneimine, I-norepinephrine, and serotonin were purchased from Sigma Chemical Co. All SP fragments and SP analogues were brought from Peninsula Laboratories, and reconsititued in 0.01 M HAc before being stored at -800C.
RESULTS
In an effort to maximize the ratio of specific to nonspecific binding of :~H-SP several preliminary experiments were performed. These data indicated that there was substantial variation in :;H-SP binding to various filters. Among the types tested GF/C glass fiber filters (Whatman) were suitable. Specific binding of zH-SP to these filters was not observed. On other preliminary experiments, it was also shown that specific '~H-SP binding was unaffected by incubation temperatures between 20"C and 39"C, or by the pH of the incubating buffer between pH 6.9 to pH 8.4. Specific binding was linear with protein concentration in the range of 200 to 1500 tLg protein per tube. HPLC analysis of 3H-SP incubated with our membrane preparation did not reveal degradation of the ligand within 60 minutes. (Data not shown). Time Course o f :JH-SP Binding to Rat Brain Membranes
Nonspecific binding of :~H-SP reached a peak within 3 minutes, however a plateau for specific and total binding was obtained only after 20 minutes of incubation. This time course was replicated using two different concentrations of '~H-SP (0.46 nM and 0.92 nM) in which exactly the same pattern of binding was observed. Specific Binding of aH-SP to Rat Brain Membranes
Specific binding reached a plateau at 4.8 nM '~H-SP. In contrast, as expected, nonspecific binding increased linearly as a function of ligand concentration. The best fitting line generated from linear regression analysis o f a Scatchard plot of the data indicated a single class of binding sites with an equilibrium dissociation constant of 0.30 nM and a density of 27.7 fmoi/mg protein. A Hill plot from the displacement curve generated with unlabeled SP was monophasic and showed no indicated for cooperatively of'~H-SP binding sites in the rat brain (Hill coefficient=0.83). Pharmacology of SP C-terminai fragments and SP related peptides to inhibit '~H-SP binding was assessed. The data in Table 1 indicates that the relative binding potencies of C-terminai fragments were parallel to the length of fragments, probably up to the hexapeptide fragment (SP 6-11) which displayed minimal inhibition of binding. The peptide fragment (SP 7-11) did not cause any noticeable inhibition. Of the two naturally occurring SP-related peptides [1], physalaemin was almost equipotent to SP, but eledoisin exhibited very low potency in displacing '~H-SP binding. SP free acid and (D-Pro", D-Trpr'Y)-SP, a suggested SP antagonist [14] also appeared to have minimal potency in competing for SP receptors. Neurotransmitters not structurally related to SP, such as I-not'epinephrine and 5-hydroxytryptamine, produced no displacement even when used at micromolar concentrations. DISCUSSION Previous studies of :)H-SP receptor binding to rat brain membrane have utilized methodologies which have left open questions concerning the binding of :~H-SP to rat brain membrane [6,21]. The data obtained in the present study indicate that the binding method used by Lee and Snyder [15] for salivary gland membranes can effectively be appfied to characterize :~H-SP binding sites in rat brain membranes. This method generated favorable ratios for specific/nonspecific binding (ranges from 6. I to 0.9).
CHARACTERISTICS OF :'H-SUBSTANCE P
835 TABLE I
INHIBITION OF 3H-SP BINDING TO RAT BRAIN BY SP FRAGMENTS AND RELATED PEPTIDES
Rat Brain Binding Relative Affinity Peptides S u b s t a n c e P (SP) SP 2-11 SP 3-11 SP 4-11 SP 5-11 SP6-11 SP 7-11 SP Free Acid Eledoisin Physalaemin (D-Pro~-Trp¢.9)-SP
Rat Brain Binding IC.~,(M)
Relative Affinity
0.76 x 10-~ ND ND 0.43 x 10-7 0.3 x 10 -s 0.93 x l0 -e i0 -a 1.0 x 10-s 0.66 x l0 -e 1.0 x l0 -9 l0 -s
1 ND ND 0.018 0.002 0.001 0.0001 0.0001 0.001 0.76 0.0001
Ref. 25 I 0.031
Ref. 3 I 0.44 0.23 0.04
0.002 0.0002 0.0001 0.008 0.16 0.0001
0.0003 0.001 0.03 0.75 0.003
Ref. 6 I 0.3 0.08 0.05 0.5 1.7 0.01 0.03 0.3 1.9
ICs,, is the concentration in which 5(~0 of 0.75 nM aH-SP was displaced from the binding sites. Values for ICs, were determined from the means of two separate experiments, each in triplicate. Two neurotransmitters, I-norepinephrine and serotonin, produced no displacement of aH-SP binding at micromolar concentrations.
The present time course study using two different 3H-SP concentrations showed that equilibrium specific binding was only reached after 20 minutes of incubation. Nakata et al. [21] did not report a time course analysis of 3H-SP receptor binding, and only incubated their tissues for 1 minute. The present results suggest that this time interval may have been insufficient to establish equilibrium binding, although difference in methodology make exact comparisons difficult. The present data indicate that there is a single class of saturable :'H-SP binding sites in rat brain membranes having a dissociation constant of 0.3 nM. These results are very similar to those obtained by Hanley et al. [6] in rat brain membranes, and by Lee and Snyder [15] in salivary gland membranes. The present data, thus, further support the hypothesis that a single class of high affinity '~H-SP binding sites exists in low density in the rat brain. These binding sites have also been shown to be noncooperative in our studies. However, some caution should be taken in this interpretation since whole brains (except cerebelli) were used in these studies. A different class of binding sites might be masked if this class of binding sites exists only in a specific region of rat brain with a very low density. So far, few attempts have been made to elucidate the relative potencies of SP fragments or SP analogues in competing for SP receptors in the mammalian CNS. Furthermore. disagreement between reports makes it difficult to determine to what extent these binding data truly reflect the biological activities of these substances in the CNS. Hanley et al. [6] argued, mainly based on the similarity between their binding data and the spinal cord depolarization action of SP and SP fragments [22], that binding sites for aH-SP described in their report reflect physiological SP receptors in the CNS. It is of particular interest to observe that the hexapeptide (SP 6-11 ) fragment appeared more potent than SP in the binding data [6]. In biological activity data from Otsuka and Konishi
[22] SP 6-11 was also demonstrated to be more potent than SP in depolarizing spinal motoneurons. However, our data revealed that SP 6-11 was only 0.1% as potent as SP at the sH-SP binding sites. This result is in agreement with the relative potency of SP 6-11 for binding to SP receptors in rat salivary gland membranes [15]. In general, the data on relative potencies of other SP fragments or SP analogues in our study is comparable to those reported by other investigators [3,25] (see Table 1), but disagrees with that of Hanley et al. [6]. At present, the reason for the apparent discrepancies between our displacement data and those of Hanley et al. [6] remains obscure. It is very interesting to note that in our data pbysalaemin exhibited almost equipotency with SP, while eledoisin appeared to be very weak in displacing 3H-SP from the binding sites since, in two biological studies, both these peptides were demonstrated to be more potent than SP. More specifically, Share et al. [27] observed a hind-limb scratching response to the intracerebral injection of SP and its related peptides into mice. In that study the effects of physalaemin and eledoisin were higher than those of SP. Similarly, these two peptides can induce more potent effects on the depolarization of spinal cord motoneurons [12]. On the other hand, other investigators [3, 16, 25] showed that, in agreement with our data, eledoisin was less potent than SP in binding to rat brain membranes. These apparent discrepancies between the potency of eledoisin in binding, and some of its biological effects may be indicative of the presence of another class of SP receptors in the CNS. These receptors might be very sensitive to eledoisin and mainly exist in the spinal cord. In fact, two types of SP binding sites in peripheral tissues, designated as SP-P and SP-E receptors, have been postulated by Sandberg and Iversen [26]. At present, it remains to be further investigated if this is also the case in the mammalian CNS.
PARK ET AL.
836 REFERENCES 1. Bertaccini, G. Active polypeptides of non-mammalian origin. Pharmacol Rev 28: 127-177, 1976. 2. Bittiger, H. Substance P in the Nervous System: Ciba Foundation Symposium 91. London: Pitman, 1982, pp. 196-200. 3. Cascierri, M. A. and T. Liang. Characterization of the substance P receptor in rat brain cortex membranes and its regulation by nucleotides. J Biol Chem 258: 5158-5164, 1983. 4. Cueilo, A. C. and I. Kanazawa. The distribution of substance P immunoreactive fibers in the rat central nervous system. J Comp Neurol 1711: 129-156, 1978. 5. Gater, P. R., C. C. Jordon, D. G. Owen. Relative activities of substance P-related peptides in the guinea-pig ileum and rat parotid gland, in vitro. Br J Pharmacol 75: 341-351, 1982. 6. Hanley, M. R., B. E. B. Sandberg, C. M. Lee, L. L. Iversen, D. E. Brundish and R. Wade. Specific binding of3H-substance P to rat brain membranes. Nature 21~: 810--812, 1980. 7. Hawcock, A. B., A. G. Hayes and M. B. Tyers. Agonist effects of (D-Pro 2, D-Phe 7, D-Trp w) substance P-evidence for different receptors. Fur J Pharmacoi m:. 135-138, 1982. 8. Henry, J. L. Effects of substance P on functionally identified units in cat spinal cord. Brain Res 114: 439--451, 1976. 9. Hokfelt, T., J. O. Kellerth, G. Nilsson and B. Pernow. Experimental immunohistochemical studies on the localization and distribution of substance P in cat primary sensory neurons. Brain Res 150:. 235--252, 1975. 10. Jessel, T. M. and L. L. lversen. Opiate analgesics inhibit substance P release from rat trigeminal nucleus. Nature 2,(~: 549551, 1977. 11. Jessel, T., A. Tsunoo, I. Kanazawa and M. Otsuka. Substance P: Depletion in the dorsal horn of rat spinal cord after section of the peripheral processes of primary, sensory neurons. Brain Res lfdl: 247-259, 1979. 12. Konishi, S. and M. Otsuka. The effects of substance P and other peptides on spinal neurons of the frog. Brain Res 65: 397-410. 1974. 13. Kronheim, S., M. C. Sheppard and B. L. Pimstone. Substance P release from rat hypothalamus and spinal cord. S A Med J 57: 827-830, 1980. 14. Leander, S., R. Hakanson, S. Rosell, K. Foikers, F. Sundler and K. Tornquist. A specific substance P antagonist blocks smooth muscle contractions induced by non-cholinergic, nonadrenergic nerve stimulation. Nature 294: 467-469, 1981. 15. Lee, C. M. and S. H. Snyder. :'H-Substance P binding to salivary gland membranes: Regulation by guanyi nucleotides and divalent cations. Mol Pharmacol 23: 563--569, 1983. 16. Lembeck, F. and G. Zetler. Substance P: a polypeptide of possible physiolollical significance, especially within the nervous system, int Rev Neurobiol 4: 159-215, 1962.
17. Liang, T. and M. A. Cascieri. Substance P stimulation of amylase release by isolated parotid cells and inhibition of substance P induction of salivation by vasoactive peptides. Mol Cell Endocrinol 15: 151-162. 1979. 18. Liang, T. and M. A. Cascieri. Substance P receptor on parotid cell membranes. J Neurosci 10: 1133-1141, 1981. 19. Ljungdahl, A., T. Hokfelt and G. Nilsson. Distribution of substance P-like immunoreactivity in the central nervous system of the rat. I. Cell bodies and nerve terminals. Neuroscience 3: 861943, 1978. 20. Lowry, O., M. Rosebrough, A. Fair and R. Randall. Protein measurement. J Biol Chem 193: 265-275, 1951. 21. Nakata, Y., Y. Kusaka, T. Segawa, H. Yajima and K. Kitagawa. Substance P: Regional distribution and specific binding to synaptic membranes in rabbit central nervous system. Life Sci 22: 259-268, 1978. 22. Otsuka, M. and S. Konishi. Substance P and excitatory transmitter of primary sensory neurons. Cold Spring Harb Symp Quan Biol 40:. 135--143, 1976, 23. Otsuka, M. and S, Knoishi. Release of substance P-like immunoreactivity from isolated spinal cord of newborn rat. Nature 2,64: 83-84, 1976. 24. Pasternak, G. W. and S. H. Snyder. Opiate receptor mechanisms. In: Neurosciences Research Program Bulletin. vol t3. edited by S. Snyder and S. Mattysee. Boston: MIT Press, 1975. p. 27. 25. Quirion, R., C. Shults, T. Moody, C. Pen, T. Chase and T. O'Donohue. Autoradiographic distribution of substance P receptors in rat central nervous system. Nature 303: 714--716, 1983. 26. Sandberg, B. E. B. and L. L. lversen. Perspective: Substance P. J Med Chem 25: 1009-1015, 1982. 27. Share, N. N. and A. Rackham. Intracerebral substance P in mice: Behavioral effects and narcotic agents. Brain Res 211: 379-386, 1981. 28. Takahashi, T. and M. Otsuka. Regional distribution of substance P in the spinal cord and nerve roots of the cat and the effect of dorsal root section. Brain Res 87: 1-11, 1975. 29. Teichberg, V. I.. S. Cohen and S. Blumberg. Distinct classes of substance P receptors revealed by a comparison of the activities of substance P and some of its segments. Regul Pept 1: 327-333. 1981. 30. Tessler, A., E. Glazer, R. Artymshyn, M. Murray and M. E. Goldberger. Recovery of substance P in the cat spinal cord after unilateral lumbosacral deafferentation. Brain Res 191: 459-470, 1980.
0196-9781/84 $3.00 + ,00
BRIEF COMMUNICATION
Characteristics of 3H-Substance P Binding Sites in Rat Brain Membranes YOUSEF
C H A N H . P A R K , * V. J O H N M A S S A R I , * R E M I Q U I R I O N , * * TIZABI,* CLIFFORD W. SHULTS* AND THOMAS L. O'DONOHUE*
*Department o f Pharmacology, College o f Medicine, Howard University 520 W. Streeet, N.W., Washington, D.C. 20059 *Experimental Therapeutics Branch. National Institute o f Neurological and Communicative Disorders and Stroke, Bethesda, MD 20205 $Section on Brain Biochemistry. Clinical Neuroscience Branch, National Institute o f Mental Health Bethesda, MID 20205 R e c e i v e d 30 J u n e 1983 PARK, C. H., V. J. MASSARI. R. QUIRION, Y. TIZABI. C. W. SHULTS AND T. L. O'DONOHUE. Characteristics of :tH-substance P binding sites in rat brain membranes. PEFTIDES 5(4) 833--836, 1984.--Binding characteristics of SHSubstance P (SP) were studied with rat brain membranes using a method applied to peripheral tissues by Lee and Snyder [15]. This method was well applicable to central nervous system (CNS) tissues. The results in the present study indicate that specific :~H-SP binding reaches a plateau only after 20 minutes of incubation, and the binding sites are saturable at a relatively low concentration of aH-SP. Scatchard analysis of specific binding data reveals a single class of binding sites with a high affinity (Kd-0.30 nM) and a low density (Bmax--27.7 fmol/mg protein) in rat brain membranes. A Hill plot of the displacement curve of :'H-SP with unlabelled SP showed no indication for cooperativity (n,=0.83). The relative potencies of binding of various SP fragments at :'H-SP binding sites were fairly parallel to the length of the C-terminal frasments. Neurotransmitters not structurally related to SP produced no effect on ~H-SP binding even when used at mieromolar concentrations. Rat brain membranes
:'H-SP binding sites
Specific binding
O N E of the important neuropeptides found in the nervous system is the undecapeptide Substance P (SP). Currently, several lines o f evidence are available to support the hypothesis that SP plays a role as a neurotransmitter or modulator in the central nervous system (CNS). as well as in the periphery. Thus, SP has an uneven distribution, [4,19], is synthesized in neurons [ 1I, 28, 30] and can be released by drugs or electrical stimuli. SP has also been shown to exert a variety o f physiological actions such as depolarization of spinal cord motoneurons, [12,22] potent vasodilation, [16] smooth muscle contraction, [29] and salivation [17] and sensory transmission in the spinal cord [8,10]. In recent years, the apparent physiological importance of SP has led investigators to attempt to characterize SP receptors in various systems including the mammalian CNS. In light of the existence of multiple receptor subtypes for some neurotransmitters and the variety o f physiological functions of SP in different systems, it is possible that multiple subtypes o f SP receptors may exist. In fact, based mainly on the disparities in the relative potencies of SP fragments and SP related peptides in various peripheral tissues, several groups have suggested that subclasses of SP receptors exist [5, 17,
18, 29]. It is not certain, however, whether this is the case within the mammalian CNS. Two groups have attempted to characterize SP receptors in the CNS by using 3H-SP as the labelled ligand in a homogenization assay [6,21]. The interpretation o f these studies is difficult, however, due to certain methodological problems encountered in the studies. Nakata et al. [21], for example, incubated tissue at 0°C for only one minute, which would probably be an insufficient time for maximal equilibrium binding o f 3H-SP to membranes. Likewise, Hanley [6] et al. employed a '~H-SP concentration for most o f their assays in which considerably higher nonspecific than specific binding was obtained. A high ratio of nonspecific to specific binding would make it difficult to detect potential low affinity binding sites. Finally, there are substantial discrepancies on the pharmacology o f 3H-SP binding sites in the mammalian CNS, depending on whether homogenization [6,21] or slice binding methodologies were employed [25]. Therefore, in this report, we have made an attempt to further explore the properties of SP receptors in the rat brain using a method applied to salivary gland membranes by Lee and Synder [15]. This method was used because it is also
833
834
PARK ET AL.
applicable to CNS tissues, and it generates a favorable ratio of specific over nonspecific binding. In the present paper, we report there is a single class of binding sites with a high affinity and a low density in rat brain membranes. These binding sites did not show cooperativity for the binding of SP. The binding site seemed highly specific for the C-terminus of SP and had a relatively high a£fnity for physalaemin but a low affinity for eledoisin. The properties of the binding sites for sH-SP appear to reflect those expected for SP receptors. METHOD
Membrane Preparation
The binding method of Lee and Synder [15] was used with a slight modification. Whole brains (except cerebelli) were removed on ice from adult male Sprague-Dawley rats and were stored at -80"C for no longer than one week. The brain tissue was homogenized in 10 vol. of ice-cold buffer A (50 mM Tris-HCl, pH=7.40, 120 raM NaCI and 5 mM KCI) with a Tissumizer (Tekmar Co.) for 20 seconds. The homogenate was centrifuged at 50,000xg for l0 minutes at 40C and the supernatant was discarded. The pellet was resuspended in ice-cold tris buffer (50 mM Tris-HCl, pH-~7.4, 300 mM KCI and 10 mM EDTA) using a sonicator before being incubated on ice for 30 minutes with gentle shaking. After centrifugation as above, the pellet was resuspended in 20 vol. of icecold buffer B (50 mM Tris-HCl, pH=7.4), centrifuged again in the same way and the supernatant was discarded. The pellet was washed in buffer B one more time repeating the same procedure as above before f'mal resuspension in 60 vol. of buffer B. Aliquots were taken for protein determination by the method of Lowry et al. [20]. Total binding was determined by adding 200/~l of final membrane suspension into tubes containing 200 ~1 of various concentrations of 3H-SP and 200 ~l of buffer containing protease inhibitors and MnCl~ (50 mM Tris-HCi, pH=7.4, 0.02% BSA, Chymostatin 0.2 rag/100 ml, Leupeptin 0.4 rag/100 mi, Bacitracin 4 rag/100 ml and 3 mM MnCI0. MnCl~ has been shown to increase specific binding of 3H-SP to peripheral tissue [15]. For the determination of nonspecific binding, unlabeled SP (prepared in the same buffer as above) was added to a final concentration of I ~M. The incubation, which was initiated by adding the membrane suspension, was performed for 20 minutes at 20°C with gentle shaking, and terminated with 5 ml of ice-cold buffer A. The tubes contents were filtered immediately through Whatman GF/C glass fiber filters under vacuum. In order to reduce nonspecific binding the filters were presoaked in distilled water containing 0.1% polyethylenimine for several hours prior to use. After the f'dtration, each of the tubes and rflters was washed once with 5 ml of ice-cold buffer A. Each filter was placed in 7 ml of Insta-gel (Packard Co.) scintillant before being counted by liquid scintillation spectrometry. Specific binding was calculated as the difference between total and nonspecific binding. MATERIALS
:~H-SP (SP) (specific activity=28.5 Ci/m Mol) was purchased from New England Nuclear at -800C until use. Chymostatin, leupeptin, bacitracin, bovine serum albumin, polethyleneimine, I-norepinephrine, and serotonin were purchased from Sigma Chemical Co. All SP fragments and SP analogues were brought from Peninsula Laboratories, and reconsititued in 0.01 M HAc before being stored at -800C.
RESULTS
In an effort to maximize the ratio of specific to nonspecific binding of :~H-SP several preliminary experiments were performed. These data indicated that there was substantial variation in :;H-SP binding to various filters. Among the types tested GF/C glass fiber filters (Whatman) were suitable. Specific binding of zH-SP to these filters was not observed. On other preliminary experiments, it was also shown that specific '~H-SP binding was unaffected by incubation temperatures between 20"C and 39"C, or by the pH of the incubating buffer between pH 6.9 to pH 8.4. Specific binding was linear with protein concentration in the range of 200 to 1500 tLg protein per tube. HPLC analysis of 3H-SP incubated with our membrane preparation did not reveal degradation of the ligand within 60 minutes. (Data not shown). Time Course o f :JH-SP Binding to Rat Brain Membranes
Nonspecific binding of :~H-SP reached a peak within 3 minutes, however a plateau for specific and total binding was obtained only after 20 minutes of incubation. This time course was replicated using two different concentrations of '~H-SP (0.46 nM and 0.92 nM) in which exactly the same pattern of binding was observed. Specific Binding of aH-SP to Rat Brain Membranes
Specific binding reached a plateau at 4.8 nM '~H-SP. In contrast, as expected, nonspecific binding increased linearly as a function of ligand concentration. The best fitting line generated from linear regression analysis o f a Scatchard plot of the data indicated a single class of binding sites with an equilibrium dissociation constant of 0.30 nM and a density of 27.7 fmoi/mg protein. A Hill plot from the displacement curve generated with unlabeled SP was monophasic and showed no indicated for cooperatively of'~H-SP binding sites in the rat brain (Hill coefficient=0.83). Pharmacology of SP C-terminai fragments and SP related peptides to inhibit '~H-SP binding was assessed. The data in Table 1 indicates that the relative binding potencies of C-terminai fragments were parallel to the length of fragments, probably up to the hexapeptide fragment (SP 6-11) which displayed minimal inhibition of binding. The peptide fragment (SP 7-11) did not cause any noticeable inhibition. Of the two naturally occurring SP-related peptides [1], physalaemin was almost equipotent to SP, but eledoisin exhibited very low potency in displacing '~H-SP binding. SP free acid and (D-Pro", D-Trpr'Y)-SP, a suggested SP antagonist [14] also appeared to have minimal potency in competing for SP receptors. Neurotransmitters not structurally related to SP, such as I-not'epinephrine and 5-hydroxytryptamine, produced no displacement even when used at micromolar concentrations. DISCUSSION Previous studies of :)H-SP receptor binding to rat brain membrane have utilized methodologies which have left open questions concerning the binding of :~H-SP to rat brain membrane [6,21]. The data obtained in the present study indicate that the binding method used by Lee and Snyder [15] for salivary gland membranes can effectively be appfied to characterize :~H-SP binding sites in rat brain membranes. This method generated favorable ratios for specific/nonspecific binding (ranges from 6. I to 0.9).
CHARACTERISTICS OF :'H-SUBSTANCE P
835 TABLE I
INHIBITION OF 3H-SP BINDING TO RAT BRAIN BY SP FRAGMENTS AND RELATED PEPTIDES
Rat Brain Binding Relative Affinity Peptides S u b s t a n c e P (SP) SP 2-11 SP 3-11 SP 4-11 SP 5-11 SP6-11 SP 7-11 SP Free Acid Eledoisin Physalaemin (D-Pro~-Trp¢.9)-SP
Rat Brain Binding IC.~,(M)
Relative Affinity
0.76 x 10-~ ND ND 0.43 x 10-7 0.3 x 10 -s 0.93 x l0 -e i0 -a 1.0 x 10-s 0.66 x l0 -e 1.0 x l0 -9 l0 -s
1 ND ND 0.018 0.002 0.001 0.0001 0.0001 0.001 0.76 0.0001
Ref. 25 I 0.031
Ref. 3 I 0.44 0.23 0.04
0.002 0.0002 0.0001 0.008 0.16 0.0001
0.0003 0.001 0.03 0.75 0.003
Ref. 6 I 0.3 0.08 0.05 0.5 1.7 0.01 0.03 0.3 1.9
ICs,, is the concentration in which 5(~0 of 0.75 nM aH-SP was displaced from the binding sites. Values for ICs, were determined from the means of two separate experiments, each in triplicate. Two neurotransmitters, I-norepinephrine and serotonin, produced no displacement of aH-SP binding at micromolar concentrations.
The present time course study using two different 3H-SP concentrations showed that equilibrium specific binding was only reached after 20 minutes of incubation. Nakata et al. [21] did not report a time course analysis of 3H-SP receptor binding, and only incubated their tissues for 1 minute. The present results suggest that this time interval may have been insufficient to establish equilibrium binding, although difference in methodology make exact comparisons difficult. The present data indicate that there is a single class of saturable :'H-SP binding sites in rat brain membranes having a dissociation constant of 0.3 nM. These results are very similar to those obtained by Hanley et al. [6] in rat brain membranes, and by Lee and Snyder [15] in salivary gland membranes. The present data, thus, further support the hypothesis that a single class of high affinity '~H-SP binding sites exists in low density in the rat brain. These binding sites have also been shown to be noncooperative in our studies. However, some caution should be taken in this interpretation since whole brains (except cerebelli) were used in these studies. A different class of binding sites might be masked if this class of binding sites exists only in a specific region of rat brain with a very low density. So far, few attempts have been made to elucidate the relative potencies of SP fragments or SP analogues in competing for SP receptors in the mammalian CNS. Furthermore. disagreement between reports makes it difficult to determine to what extent these binding data truly reflect the biological activities of these substances in the CNS. Hanley et al. [6] argued, mainly based on the similarity between their binding data and the spinal cord depolarization action of SP and SP fragments [22], that binding sites for aH-SP described in their report reflect physiological SP receptors in the CNS. It is of particular interest to observe that the hexapeptide (SP 6-11 ) fragment appeared more potent than SP in the binding data [6]. In biological activity data from Otsuka and Konishi
[22] SP 6-11 was also demonstrated to be more potent than SP in depolarizing spinal motoneurons. However, our data revealed that SP 6-11 was only 0.1% as potent as SP at the sH-SP binding sites. This result is in agreement with the relative potency of SP 6-11 for binding to SP receptors in rat salivary gland membranes [15]. In general, the data on relative potencies of other SP fragments or SP analogues in our study is comparable to those reported by other investigators [3,25] (see Table 1), but disagrees with that of Hanley et al. [6]. At present, the reason for the apparent discrepancies between our displacement data and those of Hanley et al. [6] remains obscure. It is very interesting to note that in our data pbysalaemin exhibited almost equipotency with SP, while eledoisin appeared to be very weak in displacing 3H-SP from the binding sites since, in two biological studies, both these peptides were demonstrated to be more potent than SP. More specifically, Share et al. [27] observed a hind-limb scratching response to the intracerebral injection of SP and its related peptides into mice. In that study the effects of physalaemin and eledoisin were higher than those of SP. Similarly, these two peptides can induce more potent effects on the depolarization of spinal cord motoneurons [12]. On the other hand, other investigators [3, 16, 25] showed that, in agreement with our data, eledoisin was less potent than SP in binding to rat brain membranes. These apparent discrepancies between the potency of eledoisin in binding, and some of its biological effects may be indicative of the presence of another class of SP receptors in the CNS. These receptors might be very sensitive to eledoisin and mainly exist in the spinal cord. In fact, two types of SP binding sites in peripheral tissues, designated as SP-P and SP-E receptors, have been postulated by Sandberg and Iversen [26]. At present, it remains to be further investigated if this is also the case in the mammalian CNS.
PARK ET AL.
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