Substance P Enhances Cholinergic Receptor Desensitization in a Clonal Nerve Cell Line (22Na+ Uptake/Receptor Modulation/Histrionicotoxin/Local Anesthetics) WILLIAM B

Proc. Natl. Acad. Sci. USA Vol. 77, No. 1, pp. 634-638, January 1980 Neurobiology

Substance P enhances cholinergic receptor desensitization in a clonal nerve cell line (22Na+ uptake/receptor modulation/histrionicotoxin/local anesthetics) WILLIAM B. STALLCUP AND JAMES PATRICK Developmental Biology and Neurobiology Laboratories, The Salk Institute for Biological Studies, P.O. Box 85800, San Diego, California 92138 Communicated by Robert W. Holley, October 3, 1979

ABSTRACT Substance P inhibits carbamylcholine-induced MATERIALS AND METHODS 22Na+ uptake in the clonal cell line PC12. This inhibition is noncompetitive with agonist but comretitive with Na+. Oc- The PC12 cell line is that derived by Greene and Tischler (11). tahydrohistrionicotoxin (H8-HTX) also e iibits this same pattern For the 22Na+ influx assays the cells were plated on polylys- of inhibition. Moreover, both substance P and HrHTX are very ine-coated 35-mm tissue culture dishes and used within 24 hr. effective in enhancing agonist-induced receptor desensitization. uptake stimulated chloride (car- Local anesthetics, such as QX222, also cause inhibition that is 22Na+ by carbamylcholine competitive with Na+, but they have only marginal effects on bachol) was measured at 220C as described (12-14). All ex- desensitization. Because substance P and HS-HTX cannot by periments were performed in the presence of 2 mM ouabain themselves cause desensitization, their action is dependent on to inhibit Na+ extrusion. Preincubation with ouabain was not and synergistic with the action of agonist. Furthermore, sub- found to be necessary. The 22Na+ uptake seen in the presence stance P and HS-HTX do not appear to compete for the same site of ouabain was subtracted from the overall rate found in the as QX222, which is thought to bind to the ion channel. Finally, substance P can stabilize the desensitized state of the receptor presence of both ouabain and carbachol. The initial rate of this even when added subsequent to the actual desensitization and carbachol-induced 22Na+ uptake was used in all of the data removal of agonist. Thus, substance P does not require open ion presented. Initial rates were determined by taking three time channels for binding and may modulate the activity o the re- points within the first 15 sec (e.g., at 5, 10, and 15 sec), so that ceptor-ionophore complex by binding to a distinct regulatory each data point is essentially the mean of three determinations. site. The standard deviation of these determinations is normally less Substance P produces excitatory depolarizing responses in a than 5%. In experiments that required medium with low Na+, number of different types of neurons (1-5). In addition, al- NaCl was replaced by sucrose to maintain osmolarity. though there is no evidence that substance P produces direct 22NaCl was obtained from Amersham. Most of the com- inhibition, there is evidence that substance P can block exci- pounds used in our study were generously given to us: synthetic tation produced by acetylcholine acting at nicotinic acetyl- substance P by Jean Rivier of The Salk Institute Peptide Biology choline receptors. For example, at the Mauthner fiber-giant Laboratory; [Leu]- and [Met]enkephalin by Nicholas Ling of fiber synapse in the hatchet fish (6), in cat Renshaw cells (7-9), The Salk Institute Neuroendocrinology Laboratory; bombesin, and in bovine adrenal chromaffin cells (10), activation of cho- somatostatin, neurotensin, 3-endorphin, luteinizing releasing linergic receptors is blocked by substance P. In the case of the factor, and a-melanocyte stimulating hormone by the Peptide Renshaw cells, the inhibition is not a consequence of interaction Biology and Neuroendocrinology Laboratories; local anesthetics of substance P with inhibitory glycine or y-aminobutyric acid by J. H. Steinbach of The Salk Institute Neurobiology Labo- receptors. Moreover, activation of glutamate receptors and ratory; pentobarbital by Jessxe Schulman of The Salk Institute muscarinic acetylcholine receptors is unaffected (7, 8). These Behavioral Neurobiology Laboratory; and synthetic octahy- results make it seem likely that the peptide exerts a selective drohistrionicotoxin (HS-HTX) by Palmer Taylor of the Phar- effect on nicotinic cholinergic receptors. Several alternative macology Department, University of California, San Diego, explanations might account for the observed inhibition: sub- CA. stance P might function as a cholinergic antagonist, as an ion channel blocker, or to modulate the activity of a receptor- RESULTS ionophore complex. Substance P inhibits carbachol-induced 22Na+ uptake in PC12 The PC12 cell line (11) has on its surface nicotinic acetyl- in a dose-dependent fashion. Fig. 1 shows the inhibition choline receptors that closely resemble those found on sympa- P the inhibition thetic neurons and Renshaw cells (12, 13). We have shown that achieved with substance and, for comparison, many of the functional properties of these receptors can be produced by two cholinergic antagonists, gallamine and d- investigated by using a 22Na+ influx assay to measure receptor tubocurarine; two local anesthetics, procaine and QX222; and activation (12-14). In this paper we use these cells to examine H8-HTX. Substance P and H8-HTX were each 50- to 100-fold the effects of substance P on activation of neuronal nicotinic more potent than the local anesthetics. Three other pep- acetylcholine receptors. Our goal has been to test the alternative tides-somatostatin, bombesin, and luteinizing releasing fac- ways in which substance P might produce its inhibitory effect tor-also inhibited carbachol-induced 22Na+ influx at 2- to and to compare substance P with other compounds that inhibit 5-fold higher concentrations than substance P, whereas receptor function through known mechanisms. a-melanocyte-stimulating hormone, neurotensin, /3-endorphin, and [Met]- and [Leu]enkephalin are without effect in this None The publication costs of this article were defrayed in part by page concentration range (data not shown). of the compounds charge payment. This article must therefore be hereby marked "ad- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate Abbreviations: Carbachol, carbamylcholine chloride; H8-HTX, oc- this fact. tahydrohistrionicotoxin. 634 Neurobiology: Stallcup and Patrick Proc. Natl. Acad. Sci. USA 77 (1980) 635 blockage of flux through the ion channel, or (iii) allosteric modulation of the receptor-ionophore complex. If substance P is acting as a competitive antagonist, it should be possible to overcome its effect at high concentrations of Z100 agonist. We determined the dose dependence of carbachol C 0 activation of the receptor in the presence and absence of sub- 4.80 stance P. A double reciprocal plot of this data shows that substance P behaves as a noncompetitive inhibitor of carbachol- 40 induced activation (Fig. 2A). H8-HTX also behaves in a non- 0~~~~~~~ competitive manner, but gallamine inhibits competitively, as expected of a classical cholinergic antagonist. Thus, by these criteria neither substance P nor H8-HTX interacts with the 40 agonist binding site. 20- Local anesthetics are generally thought to inhibit cholinergic function by blocking flux through the ion channel (15-17). It l-4 0 10-6 lo-, 3O0 has been postulated that the histrionicotoxins also function as [Inhibitor] ion channel blockers (18, 19), and the fact that both H8-HTX and substance P are noncompetitive with agonist suggests that FIG. 1. Inhibition of carbachol-induced 22Na+ uptake. The initial substance P could be blocking the ion channel. If these com- rate of 22Na+ uptake (receptor activity) was measured in the presence pounds block sodium influx through the ion channel, it might of 0.5 mM carbamylcholine and the indicated inhibitors. These inhibitors were tested without preincubation-i.e., they were added at be possible to overcome that blockage by increasing the external the same time as the carbachol and 22Na+. The activity of the receptor sodium concentration. The data in Fig. 2B show that the initial is expressed as the percentage of the activity found.in the absence of rate of carbachol-stimulated 22Na+ influx saturates as the so- inhibitors. 0, d-Tubocurarine; 0, substance P; 0, H8-HTX; a, pro- dium concentration is increased. Fig. 2B also shows that the caine; o, QX222; *, gallamine. inhibition achieved by substance P or H8-HTX can be overcome by increasing the sodium concentration. This behavior is also tested had any effect on 22Na+ influx when tested alone-i.e., observed in the case of QX222, but not with gallamine, which in the absence of carbachol. appears to be noncompetitive with Na+. The competitive ef- The inhibition caused by substance P might result from (i) fects of substance P, H8-HTX, and QX222 might be due to so- competitive antagonism of agonist binding to the receptor, (ii) dium competing successfully with them either for the ion inhibition of some other aspect of receptor function, such as channel or for a second site on the receptor molecule.

A B

0 x

1 2 1 2 3 4 (1/[Carbacholl) X 10-3 (1/[Na+]) X 10-' FIG. 2. Kinetic analysis of effects of inhibitors. (A) 22Na+ uptake was determined as a function of carbachol concentration. The NaCl concentration was 150 mM. When present, inhibitors were added at the same time as the carbachol and 22Na+. a, 0.2 mM gallamine; 0, 10iM substance P; 0, 10 AM H8-HTX; 3, no addition. (B) 22Na+ uptake stimulated by 0.5 mM carbachol was determined as a function of Na+ concentration. NaCl was replaced with sucrose at NaCl concentrations below 150 mM. When present, inhibitors were added at the same time as the carbachol and 22Na+. A, 0.5 mM gallamine; v, 0.2 mM QX222; 0, 3 gM substance P; 0, 2 AM H8-HTX; 0, no addition. 636 Neurobiology: Stallcup and Patrick Proc. Natl. Acad. Sci. USA 77 (1980)

a 100 A C C

ae 50 \ 50 +11 0~~~~~~ C.Oi 1 2 Preincubation time, min Recovery time, min 100 C 501

[Carbachol ]

1 2 FIG. 4. Concentration-dependence of carbachol-induced de- Preincubation time, min sensitization. PC12 cells were preincubated with various concentrations of carbachol in 10 mM Na+/2 mM Ca2+ and the rate of loss of FIG. 3. Agonist-induced desensitization and recovery. (A) PC12 activity was determined. This rate of loss is plotted in the figure as cells were preincubated at 221C in 10 mM Na+/2 mM Ca2+ with the % activity lost per sec. The rate of loss was also determined in the indicated drugs for the indicated times, washed two times with 10 mM presence of carbachol plus 10 MM substance P. In these cases, two Na+/2 mM Ca2+ buffer (requiring 30 sec), and assayed as usual in the washes were performed after the preincubation to remove substance presence of 0.5 mM carbachol. The times listed in the figure do not P. 0, Carbachol alone; 0, carbachol/10 M substance P. include the 30-sec washout period. Control cells used to establish 100%1 receptor activity were preincubated in the absence ofligands and then assayed. a, 10 AM substance P; *, 1 mM gallamine; 0, 0.5 mM car- carbachol, the rate of desensitization was dramatically in- bachol/1 mM gallamine; 0, 0.5 mM carbachol; *, 0.5 mM carba- creased. Thus, the action of substance P observed in this ex- MM chol/0.5 substance P; *, 0.5 mM carbachol/10 MM substance P. periment is dependent on the presence of agonist. A classical (B) PC12 cells were preincubated at 220C in 10 mM Na+/2 mM Ca2+ with the indicated drugs, washed twice (requiring 30 sec), and then antagonist such as gallamine does not exhibit this synergism allowed to recover in 10 mM Na+/2 mM Ca2+. The cells were assayed with agonist. In fact, as seen in Fig. 3A, gallamine inhibits at various times during the recovery period to determine receptor carbachol-induced desensitization, as might be expected be- activity. The times shown in the figure include the 30-sec washout cause it blocks agonist binding to the receptor. period. In cases in which assays were performed after 15 sec, only one Finally, concentrations of substance P (0.5 MAM) that produce wash was done. In cases in which assays were performed immediately negligible effects when tested with agonist in the 22Na+ assay after preincubation, no wash was performed; the preincubation me- (Fig. 1) are capable of increasing the desensitization achieved dium was merely replaced with the 22Na+ uptake medium. ,, 10 M substance P, 30-sec preincubation; 0, 0.5 mM carbachol, 3-min during preincubation with carbachol (Fig. SA). All of these data preincubation; *, 0.5 mM carbachol/10 MM substance P, 30-sec are consistent with the idea that substance P accelerates agon- preincubation. (C) Protocol as in A. ,, 10MuM H8-HTX; *, 0.5 mM ist-induced desensitization. However, an alternative explanation QX222; 0, 0.5 mM carbachol; 0, 0.5 mM carbachol/0.5 mM QX222; that cannot be ruled out at this point is that, in the presence of 0, 0.5 mM carbachol/10,MM H8-HTX. carbachol, substance P binds very tightly to a site (perhaps the open ion channel) from which it does not dissociate during the Burgermeister et al. (20) have suggested that HTX blocks 30-sec washout period used in Fig. 3. Thus, it would still be receptor function by enhancing agonist-induced desensitization present on the receptor when the assay is performed. of the receptor-i.e., inhibition is achieved because the ligand Fig. 3C shows that H8-HTX behaves much like substance P selects for the desensitized receptor. Desensitization of the PC12 in-this desensitization assay except that it is harder to wash out receptor can be followed by measuring the decrease in agon- and thus has some residual blocking capacity when preincu- ist-induced Na+ uptake that results from preincubation with bated by itself. In contrast to substance P and H8HTX, QX222 carbachol (Fig. 3A). In these experiments the cells were has only a small effect on desensitization. preincubated with medium containing 10 mM Na+, 2 mM The fact that the desensitized receptor has a higher affinity

Ca2 , and the indicated ligands. After various times of prein- for agonist than does the inactive or active receptor (21, 22) can cubation, the cells were washed for 30 sec in the same medium be used to help distinguish between a model in which substance minus ligands and then assayed as usual for the initial rate of P simply binds to open channels and a model in which substance carbachol-induced 22Na+ uptake. These rates were compared P binds to desensitized receptor. We have determined the to the rates observed in cells preincubated in the absence of li- concentration dependence of carbachol-induced desensitization gands. Concentrations (10 ttM) of substance P that produced and found that in the presence of substance P this dependence greater than 90% inhibition when tested with agonist in a 22Na+ is shifted by an order of magnitude to lower carbachol con- uptake assay (see Fig. 1) had no effect when cells were prein- centrations (Fig. 4), suggesting that substance P may help sta- cubated with the peptide in the absence of agonist, washed, and bilize the desensitized state of the receptor or facilitate the then assayed. It seems likely therefore that substance P is transition to this state. In other words, a concentration of car- washed out during the 30-sec wash period, and the actual ki- bachol that is incapable by itself of producing desensitization netics of recovery from preincubation with substance P (shown can have a dramatic effect when preincubated with substance in Fig. 3B) confirm this. Preincubation of cells with carbachol, P. Fig. 5A shows that H8-HTX shares this property with sub- on the other hand, produces a gradual loss of activity (Fig. 3A) stance P. On the other hand, local anesthetics and peptides other known as desensitization, which is longer lasting (see Fig. 3B). than substance P once again proved to have much less dramatic When cells were preincubated with 10,uM substance P plus effects than substance P and H8-HTX. Fig. 5B shows the ex- Neurobiology: Stallcup and Patrick Proc. Natl. Acad. Sci. USA 77 (1980) 637 B.< c 0 U 0 ,* 50 50 I4.1 0 3i U-* A B Li - i 2 Preincubation time, min FIG. 5. Comparison of effects of substance P, H8-HTX, and QX222. PC12 cells were preincubated with the following drugs at 220C for the indicated times in 10 mM Na+/2 mM Ca2 , washed twice, and assayed. (A) 0, 5 MM carbachol; v, 10 AM substance P; A, 10 AM H8-HTX; o, 10 MM substance P/5 AM carbachol; *, 10 MM H8- 3 d 6 HTX/5 MM carbachol. (B) 0, 5 MM carbachol; v, 10 MM substance Recovery time, min P; A, 0.5 mM QX222; *, 0.5 mM QX22215 MM carbachol; 03, 10 AM substance P/5 pM carbachol; A, 10MM substance P/0.5 mM QX222/5 FIG. 6. Recovery from desensitization in the presence of sub- MM carbachol. stance P. PC12 cells were desensitized for 3 min in 2 mM carbachol, washed three times in 10 mM Na+/2 mM Ca2+, and allowed to recover in the presence or absence of the indicated ligands. The beginning of ample of QX222 compared to substance P. Procaine, xylocaine, the recovery period was at the end of the third wash. After the desired times of the recovery, the cells were washed three more times and then pentobarbital, somatostatin, bombesin, and luteinizing releasing assayed. This second washout period is included in the recovery time. factor also had little or no effect on agonist-induced desensiti- 0, Recovery in either 10 mM Na'/2 mM Ca2+ or 150 mM Na+/2 mM zation. The small effect of the local anesthetics suggested a Ca2 ; o, recovery in 10 mM Na+/2 mM Ca2+/10 MM substance P;*, further experiment. If substance P binds to the same site as recovery in 150 mM Na+/2 mM Ca2+/10 MM substance P; A, effect QX222, the saturation of that site with QX222 should prevent of a 2-min incubation with substance P (10 AM) followed by three washes; *, recovery in 10 mM Na+/2 mM Ca2+/0.5 mM QX222; @, the rapid agonist-induced desensitization that occurs in the recovery in 10 mM Na+/2 mM Ca2+/0.5 mM QX222/10 MM substance presence of substance P. This prediction was not verified; in the P. presence of QX222, substance P. and carbachol the degree of desensitization was greater, not less, than that achieved by DISCUSSION carbachol and substance P. This result was also obtained when Substance P inhibits agonist-induced activation of nicotinic H8-HTX was substituted for substance P. These data suggest acetylcholine receptors on the clonal nerve line PC12. The in- that neither substance P nor H8-HTX bind to the same site as hibition achieved by substance P cannot be overcome by high QX222. concentrations of agonist but can be overcome by high con- Having established that the effect of substance P (and H8- centrations of sodium. H8-HTX and QX222 share these two HTX) in these preincubation experiments is dependent on the properties with substance P. Thus, these compounds do not act presence of agonist, we can consider an additional question. as classical antagonists but rather as compounds that modulate Does the ability of substance P to trap receptor in the desensi- receptor activity. tized state require that Both substance P and H8-HTX increase the rate of agonist- substance P be present during receptor induced receptor desensitization. Enhanced desensitization was activation? Fig. 6 shows an experiment in which PC12 cells proposed as a possible mechanism of action for substance P in were substantially desensitized with carbachol so that the re- the case of cat Renshaw cells (7) and for HTX in the case of ceptor state changed from inactive to active to desensitized. The chicken muscle (20). In PC12 the synergism between substance carbachol was then washed out and the recovery of activity was P and carbachol in producing receptor desensitization is dem- monitored in the presence and in the absence of substance P. onstrated in two experiments. In one case concentrations of When these experiments were performed in 10 mM Na+, substance P that produce negligible effects when tested with substance P significantly slowed the recovery from the de- agonist in the sodium uptake assay do cause inhibition when sensitized state, suggesting that substance P can bind to and preincubated with cells in the presence of carbachol. Secondly, stabilize the desensitized state of the receptor and does not re- the concentration of carbachol required to produce desensiti- quire the active state of the receptor or open ion channels for zation is decreased to 1/10th in the presence of substance P. binding. When the same experiments were performed in 150 These properties are shared with HS-HTX. Thus, substance P mM Na+, the effect of substance P on recovery was decreased. appears to function by trapping the receptor in the desensitized This is reminiscent of the competition observed earlier between state rather than by simply binding to open receptor channels substance P and Na+. In contrast, QX222 had little effect on and blocking ion flux. This hypothesis is strengthened by the the rate of recovery, regardless of the Na+ concentration. finding that receptors that are driven to the desensitized state Moreover, the presence of QX222 did not lessen the effect of by prolonged incubation in carbachol in the absence of sub- substance P on recovery (in 10 mM Na+), suggesting once again stance P recover from desensitization more slowly in the pres- that these two ence of substance P than in its absence. Thus, substance P does ligands do not have a common site of action. not have to be present during the activation process. These same types of experiments could not be performed with Changeux and coworkers (23) have proposed that local an- H8-HTX because of the difficulty encountered in washing out esthetics, besides binding to and blocking open ion channels, the drug (see Figs. 3C and 5A). The lingering inhibition caused can also enhance agonist-induced desensitization. This is by H8-HTX alone obscures the small effects we are looking compatible with a model in which the receptor and ion channel for. are allosterically coupled. In addition, there have been reports 638 Neurobiology: Stallcup and Patrick Proc. Natl. Acad. Sci. USA 77 (1980) suggesting that the site of action of HTX is also the ion channel cholinergic excitation of Renshaw cells (28), although the effect (18, 19, 24). By analogy, substance P might also have such a dual appeared less pronounced than that of substance P (9).) This inhibitory function: blocking the ion channel and in doing so does not preclude the exciting possibility that these other pep- increasing the rate of receptor desensitization. The competition tides may have modulatory functions at other types of receptors observed between substance P and Na+ is consistent with this in the nervous system, such as the modulation of glutamate- sort of model. Nevertheless, there are several pieces of infor- induced excitation of spinal neurons by [Leulenkephalin (29) mation that are inconsistent with this model. The first is the and substance P (30). observation that substance P and H8-HTX are much more effective in enhancing agonist-induced desensitization than are We especially thank Dr. Jean Rivier and Dr. Palmer Taylor for local anesthetics or other peptides such as somatostatin, bom- supplying us with substance P and H8-HTX, respectively, and Dr. J. H. Steinbach for many helpful discussions. This work was supported besin, and luteinizing releasing factor. The second is that, in the by National Institutes of Health Grant NS21781-01 and Sloan Foun- experiment shown in Figs. 5B and 6, QX222 does not seem to dation Fellowship BR-1873 to W. B. S. and by National Institutes of compete with substance P. The third is that, unlike QX222, Health Grant NS13546-02 and a Muscular Dystrophy Association, Inc. substance P can stabilize the desensitized state of the receptor Grant to J.P. even when no ion channels are open, and in doing so still appears to compete with Na+ (Fig. 6). Thus, the possibility exists 1. Konishi, S. & Otsuka, M. (1974) Nature (London) 252, 734- that substance P and HTX act at a modulatory site that is dis- 735. 2. Henry, J. L., Krnjevic, K. & Morris, M. E. (1975) Can. J. Physiol. tinct from the ion channel. Pharmacol. 53, 423-432. In this regard it is important to note that HTX exhibits some 3. Krnjevic, K. & Morris, M. E. (1974) Can. J. Physiol. Pharmacol. differences from local anesthetics in voltage-clamp experiments 52, 736-744. in which the rate of decay of the end plate current is measured. 4. Phillips, J. W. & Limacher, J. J. (1974) Brain Res. 69, 158- Local anesthetics produce a characteristic biphasic decay curve 163. (15, 16, 25)-an initial decay more rapid than normal, caused 5. Leeman, S. & Mroz, E. A. (1974) Life Sci. 15,2033-2044. by blocking of the ion channels, followed by a very slow decay, 6. Steinacker, A. & Highstein, S. M. (1976) Brain Res 114, 128- caused by slow unblocking of the channels and subsequent 133. closing of the channels (Model 1). 7. Belcher, G. & Ryall, R. W. (1977) J. Physiol. 272, 105-119. 8. Krnjevic, K. & Lekic, D. (1977) Can. J. Physiol. Pharmacol. 55, (Model 1) Closed >± Open >. Blocked 958-961. 9. Davies, J. & Dray, A. (1977) Nature (London) 268,351-352. HTX produces only the first part of this phenomenon-a decay 10. Livett, B. G., Kozousek, V., Mizobe, F. & Dean, D. M. (1979) that is more rapid than usual but that nevertheless still fits a Nature (London) 278, 256-257. single exponential (19, 26). Thus, the return to the normal 11. Greene, L. A. & Tischler, A. S. (1976) Proc. Natl. Acad. Sci. USA resting state from the inactive state apparently does not proceed 73, 2424-2428. through an open channel state (Model 2). This model resembles 12. Patrick, J. & Stallcup, W. B. (1977) Proc. Natl. Acad. Sci. USA 74, 4689-4692. a simplified scheme for desensitization. 13. Patrick, J. & Stallcup, W. B. (1977) J. Biol. Chem. 252, 8629- Closed - Open 8633. (Model 2) 14. Stallcup, W. B. (1979) J. Physiol. 286,525-540. 15. Ruff, R. L. (1977) J. Physiol. 264,89-124. Inactive 16. Adams, P. R. (1977) J. Physiol. 268,271-289. 17. Neher, E. & Steinbach, J. H. (1978) J. Physiol. 277, 153-176. It is also worthwhile to recall the existence of the metaphilic 18. Albuquerque, E. X., Barnard, E. H., Chiu, T. H., Lapa, A. J., antagonists studied by Rang and Ritter (27). Decamethonium Dolly, J. O., Jansson, S., Daly, J. & Witkop, B. (1973)'Proc. Natl. derivatives thought to bind at or near the agonist binding site, Acad. Sci. USA 70,949-953. not to the ion channel, were found to enhance agonist-induced 19. Eldefrawi, A. T., Eldefrawi, M. E., Albuquerque, E. X., Oliveira, desensitization. A. C., Mansour, N., Adler, M., Daly, J. W., Brown, G. B., Bur- In summary, we have shown that the ability of substance P germeister, W. & Witkop, B. (1977) Proc. Natl. Acad. Sci. USA to block acetylcholine receptor function in PC12 rests largely 74,2172-2176. We have not 20. Burgermeister, W., Catterall, W. A. & Witkop, B. (1977) Proc. in its capacity to enhance receptor desensitization. Natl. Acad. Sci. USA 74,5754-5758. yet been able to determine unequivocally whether substance 21. Weber, M., David-Pfeuty, T. & Changeux, J.-P. (1975) Proc. Nati. P exerts this effect by binding to the ion channel or to some Acad. Sci. USA 72,3443-3447. other anionic site. We have, however, obtained data that is not 22. Weiland, G., Georgia, B., Wee, V. T., Chignell, C. F. & Taylor, consistent with substance P binding either to an open ion P. (1976) Mol. Pharmacol. 12, 1091-1105. channel, or to a resting, closed ion channel, or to the same site 23. Cohen, J. B., Weber, M. & Changeux, J.-P. (1974) Mol. Phar- that binds the local anesthetic QX222. Thus, the idea that macol. 10, 904-932. substance P binds to an anionic modulatory site remains plau- 24. Kato, G. & Changeux, J.-P. (1975) Mol. Pharmacol. 12, 92- sible. 100. The other we tested did not exhibit the effects 25. Steinbach, A. B. (1968) J. Gen. Physiol. 52, 144-161. neuropeptides 26. Albuquerque, E. X., Kuba, K. & Daly, J. (1974) J. Pharmacol. shown by substance P. At concentrations above 10 ,M, soma- Exp. Ther. 189, 513-524. tostatin, bombesin, and luteinizing releasing factor gave partial 27. Rang, H. P. & Ritter, J. M. (1969) Mol. Pharmacol. 5, 394- blockage of carbachol-stimulated 22Na+ uptake but did so 411. without any effect on agonist-induced desensitization, whereas 28. Davies, J. & Dray, A. (1976) Nature (London) 262, 603-604. a-melanocyte stimulating hormone, neurotensin, f-endorphin, 29. Barker, J. L., Neale, J. H., Smith, T. G. & MacDonald, R. L. (1978) and [Leu]- and [Met]enkephalin failed to block at all. (It should Science 199, 1451-1453. be noted that [Metlenkephalin has been reported to suppress 30. Vincent, J.-D. & Barker, J. L. (1979) Science 205, 1409-1412.