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Differences between rats and guinea pigs in gastrointestinal and nervous system substance P levels
Neuropeptides 1:
383-389, 1981
DIFFERENCES BET-tiEEN RATS AND GUINEA PIGS IN GASTROINTESTINAL AND NERVOUS SYSTEM SUBSTANCE P LEVELS S.H. Buck, P.P. Deshmukh, H.I. Yamamura, and T.F. Burks Department of Pharmacology, College of Medicine, University of Arizona Health Sciences Center, Tucson, AZ 85724 (reprint requests to SHB) ABSTRACT Substance P imnunoreactivity was measured in regions of the gastrointestinal and nervous systems of age-matched adult rats and guinea pigs. Guinea pigs were found to have substantially higher substance P levels than rats in the gastrointestinal system, in dorsal roots and ganglia, and in dorsal spinal cord. Rats had higher levels in hypothalamus while levels in corpus striatum and ventral spinal cord were comparable in both species. Treatment oE guinea pigs with parenteral capsaicin reduced substance P levels only in dorsal roots and ganglia and in dorsal spinal cord. INTRODUCTION The undecapeptide, substance P (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-GlyLeu-Met-NH2), was first isolated by von Euler and Gaddum (1) in alcoholic extracts of brain and gut. Extracts containing the peptide were extremely potent in causing contraction of isolated intestinal tissue and in lowering blood pressure in anesthetized animals (1,2). In 1970, Chang and Leeman unexpectedly isolated sufficient quantities of substance P from bovine hypothslami to allow subsequent characterization of its structure (3,4). Leeman's group then proceeded to synthesize the peptide and to develop a radioimmunoassay for substance P (5,6). Substance P occurs in organisms at all levels of the phylogenetic tree (7) and there is evidence that the peptide has a neurophysiological role in the mammalian gastrointestinal system (for review see 8) and central nervous system (for review see 9). Evidence is particularly compelling for a role of the peptide as a neurotransmitter or neuromodulator in some primary afferent neurons mediating nociceptive transmission (IO). Prior to the development of a radioimmunoassav for substance P, quantitation of the peptide in tissues was accomplished by bioassay. In vitro gastrointestinal tissue preparations were frequently employed and the Finea pig ileum was a commonly used bioassay system because of its sensitivity (7). It is now known that authentic substance P is one of the most potent agents in 383
contracting guinea pig ileum with two groups reporting an identical ED50 valjw Eor the peptide oE 2.5 X 10e9 M (11,121. In the isolated guinea pig ileum, the molar potency of substance P is 40 times greater than that of acetylcholine, 168 times greater than that of histamine, and 300 times greater than that of 5-hydroxytryptamine (12). Although early bioassay determinations suggested similar levels of the peptide in gut of rat and guinea pig (7), gastrointestinal tissue of the rat is considerably less sensitive to the contractile effects of substance p than that of guinea pigs (3,5). There also appeared to be little difference between the two species in brain levels of substance P (7). We have used an antiserum to substance P for comparison by radioimmunoassay of levels of the peptide in gastrointestinal and nervous tissue of the rat and the guinea pig. The effects of the putative substance neurotoxin, capsaicin (13,14), on substance P levels were determined in the guinea pig. METHODS For the guinea pig and rat comparison experiment, age-matched male Hartley guinea pigs (447-577 g; 10 l/2 weeks old) and male Sprague-Dawley rats (295-330 g; 10 l/2 weeks old) were used to eliminate differences in substance P levels due to age (15). Tissues from the two species were assayed at the same time. In the capsaicin experiment, young male Hartley guinea pigs (233-265 g) were employed. Capsaicin was injected subcutaneously at the back of the neck in a vehicle consisting of 10% ethanol-10% TWEEN 80-80% saline (v:v:v). Control animals received the vehicle alone. Immediately after the first capsaicin injection, guinea pigs were placed for a short time in a chamber gassed with 95% 02:5% CO2 and a mist of 0.75% isoproterenol to prevent fatalities from capsaicin-induced bronchospasms. For the radioimmunoassay of substance P, animals were decapitated and tissues were removed and placed on a chilled surface for dissection. Four to six lumbar dorsal roots plus attached ganglia from each animal were pooled. The corresponding lumbar spinal cord was hemisected while frozen into dorsal and ventral parts at the level of the central canal. All tissues were frozen on dry ice and weighed frozen. Tissues were stored at -8OOC until time of assay. Previous studies have shown substance P to be extremely stable in post-mortem tissues (16,17,18). For the radioimmunoassay, tissues were homogenized in lo-20 volumes of ice cold 2 11 acetic acid and centrifuged. Extraction of substance P was quantitative. The supernatant was lyophilized and re-dissolved in buEfer at the time of assay. An equilibrium radioimmunoassay was performed using a 1:50,000 dilution of an antiserum produced in rabbits and 1251-tyrS -substance P prepared by the chloramine T method to a specific activity of approximately 2000 Ci/mmole as tracer (19). Sensitivity of the assay was 7 fmole substance P in 100 ul of sample. Assay of varying dilutions of tissue samples produced displacement curves that were parallel to the displacement curve for authentic substance P. The anti-substance P serum exhibited the following approximate cross-reactivities: 1% with physalaemin, 0.01% with eledoisin, 0.001% with somatostatin, 0.0001% with neurotensin, and less than 0.00001% with bombesin, bradykinin, vasoactive intestinal polypeptide, pentagastrin, CCK (27-33), thyrotropin-releasing hormone , angiotensin II, a-endorphin, S-endorphin, yendorphin, leu5-b-endorphin, met-enkephalin, D-ala2-leu-enkephalinamide, dynorphin-(l-13) and des-tyr-y-endorphin. 384
P
Capsaicin was obtained from Sigma Chemical Co. (St. Louis, Missouri). Peptides were obtained from Vega Biochemicals (Tucson, Arizona), Peninsula Laboratories (San Carlos, California), or Beckman Bioproducts (Palo Alto, California). RESULTS Using an antiserum with considerable specificity for substance P, we have observed striking differences in levels of the peptide in rats and guinea pigs. The data in Table I indicate that guinea pigs have significantly higher substance P levels than rats in duodenum, ileum, and colon. Guinea pig ileum contained approximately 2 l/2-fold higher levels while duodenum and colon of guinea pigs each contained nearly lo-fold higher levels of substance P than comparable tissues from rats. In nervous tissue, guinea pig corpus striatum contained equivalent levels of substance P to rat corpus striatum while rat hypothalamus contained 60% more substance P than guinea pig hypothalamus. At the spinal level, guinea pigs had 4-fold higher peptide levels in dorsal roots and ganglia and over P-fold higher levels in dorsal spinal cord. Substance P levels in ventral spinal cord were similar in both species (Table I).
TABLE I Comparison of substance P levels in rats and guinea pigs. pmole/g wet weight of tissue with mean -+ s.e.m. shown. Rat (n = 6) Duodenum Ileum Colon Hypothalamus Corpus striatum Dorsal root plus ganglion Dorsal cord Ventral cord
Values are
Guinea Pig (n = 6)
P
7.0 + 2.4 19.1 + 4.3 5.0 z 0.7
50.6 + 3.5 45.1 5 5.3 46.5 5 - 3.5
c.001 c.01 c.001
131.6 + 7.1 29.0 + 2.3 3.3 5 0.6
81.4 + 7.8 34.0 T 3.0 13.6 - 2.7
c.001 NS c.01
317.5 + 13.7 17.4 5 - 2.5
c.001 NS
141.6 + 10.4 12.4 - 1.6
The putative substance P neurotoxin, capsaicin, has been reported to deplete the peptide from primary afferent fibers in adult rats (13,141. Parenteral administration of high doses of capsaicin to guinea pigs produced a reduction in substance P levels that was limited to dorsal roots plus ganglia and dorsal spinal cord (Table II). The reduction was 78% in dorsal roots and 43% in spinal cord. There were no significant changes in gastrointestinal tissue or in brain corpus striatum or hypothalamus.
385
TABLE II Effect of parenteral capsaicin administration on substance P levels in guinea pigs. Values are pmole/g wet weight of tissue with mean 2 s.e.m. shown. Capsaicin was administered in consecutive daily doses of 50,200,200,400,400 mg/kg S.C. and animals were killed 8 days after the last injection. Capsaicin (n = 4)
Control (n = 5) Duodenum Ileum Colon Hypothalamus Corpus striatum Dorsal root plus ganglion Dorsal cord Ventral cord
P
93.0 + 18.3 73.9 5 12.6 65.4 E 4.6
112.1 + 11.4 115.8 5 23.4 81.1 z 5.1
NS NS NS
129.9 + 24.3 56.1 + 5.1 24.6 ;: 6.0
149.9 + 32.2 95.3 7 38.1 5.4 5 0.5
NS NS <.025
291.5 + 36.4 17.1 + 3.0
165.6 + 25.1 16.8 i 5.0
c.025 NS
DISCUSSION Our results clearly show that gastrointestinal tissues of guinea pigs contain substantially higher levels of substance P than similar tissues from rats. This is in contrast to early bioassay determinations which suggested that the two species had similar levels in gut tissue (7). The bioassay results, however, are extremely equivocal since early experimenters used impure tissue extracts containing many gastrointestinally active agents (20). Hokfelt et al. (21) have reported no species difference in number of substance P cell bodies in colchicine-treated gut tissues from rats and guinea pigs. However, even though there are definite intrinsic substance P-containing neurons in the gut (22,231, there is also evidence for extrinsic substance P innervation of the gastrointestinal system (23,24). Species differences in this extrinsic innervation, undetectable by counting of peptide cell bodies in gut tissue, could account for the differences in substance P levels we observed between rat and guinea pig intestinal tissues. In support of this possibility is the observation by Hokfelt et al. (25) that mesenteric ganglia of guinea pigs contain more substance P immunofluorescent fibers than ganglia from rats. In the central nervous systems of the two species, there were similarities and differences in substance P levels. While levels were comparable in corpus striatum of rats and guinea pigs, rats had significantly higher levels of the peptide in the hypothalamus. Whether the higher levels in rat hypothalamus result from more substance P-containing neurons intrinsic to the hypothalamus or from more peptide-containing afferents to the hypothalamus remains to be determined. Substance P levels were similar in ventral spinal cord from both species whereas guinea pigs had over P-fold higher peptide levels than rats ir dorsal cord. That the higher levels in guinea pig dorsal cord result from 386
substance P-containing somatic primary afferents is suggested by the 4-fold higher peptide levels in guinea pig dorsal roots compared to rats. This finding indicates either that guinea pigs have a greater number of substance P-containing primary afferent neurons or that the substance P-containing neurons present in guinea pigs contain higher levels of the peptide compared Greater numbers of substance P immunopositive fibers in to those of rats. guinea pig mesenteric ganglia compared to ganglia of rats and the suggestion that some of these originate from neurons in spinal sensory ganglia support the former of these two possibilities (25,26). Capsaicin, the major irritant in hot peppers of the plant genus Capsicum, has recently been reported to produce a depletion of substance P from primary afferents upon parenteral administration to adult rats (13,141. The results of the present study indicate that capsaicin has a similar neurotoxic action in adult guinea pigs. Substance P levels were reduced by the agent extensively in dorsal roots and moderately in dorsal spinal cord. There were no changes in levels of the peptide in the two brain regions studied or in any The reductions in substance P levels in part of the gastrointestinal tract. guinea pig afferents were similar in magnitude to those observed in rats (13; Buck et al., unpublished data). Thus, despite the marked differences in substance P levels in primary afferent neuronal tissues of rats and guinea pigs, peptide-containing afferents in the two species exhibit at least a qualitatively similar susceptibility to the effects of capsaicin. The possibility that there is a difference between the two species in the structure of substance P in the immunoreactive region of the molecule cannot be unequivocally ruled out as the reason for our results. However, non-immunological analytical methods employed by others have not detected species differences in peptide structure. Substantially higher levels of substance P in guinea pig gastrointestinal and primary afferent tissue may indicate a more important neurophysiological role of the peptide in these systems of the guinea pig compared to the rat. The greater sensitivity of guinea pig gut tissue to the contractile effects of substance P supports this contention for guinea pig gastrointestinal function (3,5). Similarly, we have observed that capsaicin treatment of adult guinea pigs results in a profound thermal analgesia, an effect not seen in adult rats treated with capsaicin (14; Buck et al., submitted). In toto, these results suggest that the guinea pig may be a more useful species than the rat in which to investigate the biological roles of substance P and the physiological and pharmacological changes consequent to capsaicin neurotoxicity. ACKNOWLEDGEMENTS I___ Appreciation is extended to Dr. Marvin R. Brown, The Salk Institute, for generous supplies of substance P antiserum. Supported by USPHS grants DA02163, NS15420, and MH30626. H.1. Yamamura is a recipient of an RSDA, Type 11 (EIH-00095), from the National Institute of Mental Health.
387
REFERENCES :-_1. von Euler US, Gaddum JH.
certain tissue extracts.
(1931) An unidentified depressor substance in J. Physiol. (London) 72, 74-87.
2. Gaddum JH, Schild H. (1934) Depressor substances in extracts of intestine. J. Physiol. (London) 83, 1-14. 3. Chang MM, Leeman SE. (1970) Isolation of a sialogogic peptide from bovine hypothalamic tissue and its characterization as substance P. J. Biol. Chem. 245, 4784-4790. 4. Chang MM, Leeman SE, Niall HD. (1971) Amino-acid sequence of substance P. Nature New Biology 232, 86-87. 5. Tregear GW, Niall HD, Potts JT et al. (1971) Nature New Biology 232, 87-89.
Synthesis of substance P.
6. Powell D, Leeman S, Tregear GW et al. (1973) Radioimmunoassay for substance P. Nature New Biology 241, 252-254. 7. Lembeck F, Zetler G. (1962) Substance P: a polypeptide of possible physiological significance, especially within the nervous system. Inter. Rev. Neurobiol. 4, 159-215. a.
Bury RW, Mashford ML. (1977) Substance P: its pharmacology and physiological roles. Aust. J. Exp. Biol. Med. Sci. 55, 671-735.
9.
Nicoll RA, Schenker C, Leeman SE. (1980) Substance P as a transmitter candidate. Ann. Rev. Neurosci. 3, 227-268.
10.
Otsuka M, Takahashi T. (1977) Putative peptide neurotransmitters. Ann. Rev. Pharmacol. 17, 425-439.
11.
Bury RW, Mashford ML. (1976) Biological activity of C-terminal partial sequences of substance P. J. Med. Chem. 19, 854-856.
12. Rose11 S, Bjorkroth U, Chang D et al. (1977) Effects of substance P and analogs on isolated guinea pig ileum. In Substance P (ed US von Euler and B Pernow), pp. 83-88. Raven Press, New York. 13. Jesse11 TM, Iverson LL, Cue110 AC. (1978) Capsaicin-induced depletion of substance P from primary sensory neurons. Brain Research 152, 183-188. 14. Hayes AG, Tyers MB. (1980) Effects of capsaicin on nociceptive heat, pressure, and chemical thresholds and on substance P levels in the rat. Brain Research 189, 561-564. 15. Gilbert RFT, Emson PC. (1979) Substance P in rat CNS and duodenum during development. Brain Research 171, 166-170. 16. Gale JS, Bird ED, Spokes EG et al. (1978) Human brain substance P: distribution in controls and Huntington's chorea. J. Neurochem. 30, 633-634.
388
17. Kanazawa I, Jesse11 T. (1976) Post mortem changes and regional distribution of substance P in the rat and mouse nervous system. Research 117, 362-367.
Brain
(1980) The development and application 18. Lee CM, Emson PC, Iversen LL. Life Sciences 27, a novel N-terminal directed substance P antiserum. 535-543.
of
(1979) Substance P. In Methods of Hormone 19. Mroz EA, Leeman SE. Radioimmunoassay (ed BM Jaffee and HR Behrman), pp 121-137. Academic Press, New York. 20. Skrabanek P, Powell D. (1977) Substance P: Reviews. Eden Press, St. Albans, Vermont.
volume 1, Annual Research
21. Schultzberg M, Hokfelt T, Nilsson G et al. (1980) Distribution of peptide and catecholamine-containing neurons in the gastrointestinal tract immunohistochemical studies with antisera to of rat and guinea pig: substance P, vasoactive intestinal polypeptide, enkephalins, somatostatin, gastrin/cholecystokinin, neurotensin, and dopamine-B-hydroxylase. Neuroscience 5, 689-744. 22. Schultzberg M, Dreyfus CF, Gershon MD et al. (1978) VIP-, enkephalin-, substance P-, and somatostatin-like immunoreactivity in neurons intrinsic to the intestine: immunohistochemical evidence from organotypic tissue cultures. Brain Research 155, 239-248. 23. Hokfelt T, Kellerth J-O, Nilsson G, Pernow B. (1975) Experimental inrmunohistochemical studies on the localization and distribution of Brain Research 100, 235-252. substance P in cat primary sensory neurons. 24. Hokfelt T, Elde R, Johansson 0 et al. (1976) Immunohistochemical evidence for separate populations of somatostatin-containing and substance Neuroscience 1, 131-136. P-containing primary afferent neurons. 25. Hokfelt T, Elfvin M, Schultzberg M et al. (1977) On the occurrence of substance P-containing fibers in sympathetic ganglia: immunohistochemical evidence. Brain Research 132, 29-41. 26. Hokfelt, T., Johansson, O., Ljungdahl, A et al. (1980) Peptidergic neurons. Nature 284, 515-521.
Accepted
3 March,
1981
389
383-389, 1981
DIFFERENCES BET-tiEEN RATS AND GUINEA PIGS IN GASTROINTESTINAL AND NERVOUS SYSTEM SUBSTANCE P LEVELS S.H. Buck, P.P. Deshmukh, H.I. Yamamura, and T.F. Burks Department of Pharmacology, College of Medicine, University of Arizona Health Sciences Center, Tucson, AZ 85724 (reprint requests to SHB) ABSTRACT Substance P imnunoreactivity was measured in regions of the gastrointestinal and nervous systems of age-matched adult rats and guinea pigs. Guinea pigs were found to have substantially higher substance P levels than rats in the gastrointestinal system, in dorsal roots and ganglia, and in dorsal spinal cord. Rats had higher levels in hypothalamus while levels in corpus striatum and ventral spinal cord were comparable in both species. Treatment oE guinea pigs with parenteral capsaicin reduced substance P levels only in dorsal roots and ganglia and in dorsal spinal cord. INTRODUCTION The undecapeptide, substance P (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-GlyLeu-Met-NH2), was first isolated by von Euler and Gaddum (1) in alcoholic extracts of brain and gut. Extracts containing the peptide were extremely potent in causing contraction of isolated intestinal tissue and in lowering blood pressure in anesthetized animals (1,2). In 1970, Chang and Leeman unexpectedly isolated sufficient quantities of substance P from bovine hypothslami to allow subsequent characterization of its structure (3,4). Leeman's group then proceeded to synthesize the peptide and to develop a radioimmunoassay for substance P (5,6). Substance P occurs in organisms at all levels of the phylogenetic tree (7) and there is evidence that the peptide has a neurophysiological role in the mammalian gastrointestinal system (for review see 8) and central nervous system (for review see 9). Evidence is particularly compelling for a role of the peptide as a neurotransmitter or neuromodulator in some primary afferent neurons mediating nociceptive transmission (IO). Prior to the development of a radioimmunoassav for substance P, quantitation of the peptide in tissues was accomplished by bioassay. In vitro gastrointestinal tissue preparations were frequently employed and the Finea pig ileum was a commonly used bioassay system because of its sensitivity (7). It is now known that authentic substance P is one of the most potent agents in 383
contracting guinea pig ileum with two groups reporting an identical ED50 valjw Eor the peptide oE 2.5 X 10e9 M (11,121. In the isolated guinea pig ileum, the molar potency of substance P is 40 times greater than that of acetylcholine, 168 times greater than that of histamine, and 300 times greater than that of 5-hydroxytryptamine (12). Although early bioassay determinations suggested similar levels of the peptide in gut of rat and guinea pig (7), gastrointestinal tissue of the rat is considerably less sensitive to the contractile effects of substance p than that of guinea pigs (3,5). There also appeared to be little difference between the two species in brain levels of substance P (7). We have used an antiserum to substance P for comparison by radioimmunoassay of levels of the peptide in gastrointestinal and nervous tissue of the rat and the guinea pig. The effects of the putative substance neurotoxin, capsaicin (13,14), on substance P levels were determined in the guinea pig. METHODS For the guinea pig and rat comparison experiment, age-matched male Hartley guinea pigs (447-577 g; 10 l/2 weeks old) and male Sprague-Dawley rats (295-330 g; 10 l/2 weeks old) were used to eliminate differences in substance P levels due to age (15). Tissues from the two species were assayed at the same time. In the capsaicin experiment, young male Hartley guinea pigs (233-265 g) were employed. Capsaicin was injected subcutaneously at the back of the neck in a vehicle consisting of 10% ethanol-10% TWEEN 80-80% saline (v:v:v). Control animals received the vehicle alone. Immediately after the first capsaicin injection, guinea pigs were placed for a short time in a chamber gassed with 95% 02:5% CO2 and a mist of 0.75% isoproterenol to prevent fatalities from capsaicin-induced bronchospasms. For the radioimmunoassay of substance P, animals were decapitated and tissues were removed and placed on a chilled surface for dissection. Four to six lumbar dorsal roots plus attached ganglia from each animal were pooled. The corresponding lumbar spinal cord was hemisected while frozen into dorsal and ventral parts at the level of the central canal. All tissues were frozen on dry ice and weighed frozen. Tissues were stored at -8OOC until time of assay. Previous studies have shown substance P to be extremely stable in post-mortem tissues (16,17,18). For the radioimmunoassay, tissues were homogenized in lo-20 volumes of ice cold 2 11 acetic acid and centrifuged. Extraction of substance P was quantitative. The supernatant was lyophilized and re-dissolved in buEfer at the time of assay. An equilibrium radioimmunoassay was performed using a 1:50,000 dilution of an antiserum produced in rabbits and 1251-tyrS -substance P prepared by the chloramine T method to a specific activity of approximately 2000 Ci/mmole as tracer (19). Sensitivity of the assay was 7 fmole substance P in 100 ul of sample. Assay of varying dilutions of tissue samples produced displacement curves that were parallel to the displacement curve for authentic substance P. The anti-substance P serum exhibited the following approximate cross-reactivities: 1% with physalaemin, 0.01% with eledoisin, 0.001% with somatostatin, 0.0001% with neurotensin, and less than 0.00001% with bombesin, bradykinin, vasoactive intestinal polypeptide, pentagastrin, CCK (27-33), thyrotropin-releasing hormone , angiotensin II, a-endorphin, S-endorphin, yendorphin, leu5-b-endorphin, met-enkephalin, D-ala2-leu-enkephalinamide, dynorphin-(l-13) and des-tyr-y-endorphin. 384
P
Capsaicin was obtained from Sigma Chemical Co. (St. Louis, Missouri). Peptides were obtained from Vega Biochemicals (Tucson, Arizona), Peninsula Laboratories (San Carlos, California), or Beckman Bioproducts (Palo Alto, California). RESULTS Using an antiserum with considerable specificity for substance P, we have observed striking differences in levels of the peptide in rats and guinea pigs. The data in Table I indicate that guinea pigs have significantly higher substance P levels than rats in duodenum, ileum, and colon. Guinea pig ileum contained approximately 2 l/2-fold higher levels while duodenum and colon of guinea pigs each contained nearly lo-fold higher levels of substance P than comparable tissues from rats. In nervous tissue, guinea pig corpus striatum contained equivalent levels of substance P to rat corpus striatum while rat hypothalamus contained 60% more substance P than guinea pig hypothalamus. At the spinal level, guinea pigs had 4-fold higher peptide levels in dorsal roots and ganglia and over P-fold higher levels in dorsal spinal cord. Substance P levels in ventral spinal cord were similar in both species (Table I).
TABLE I Comparison of substance P levels in rats and guinea pigs. pmole/g wet weight of tissue with mean -+ s.e.m. shown. Rat (n = 6) Duodenum Ileum Colon Hypothalamus Corpus striatum Dorsal root plus ganglion Dorsal cord Ventral cord
Values are
Guinea Pig (n = 6)
P
7.0 + 2.4 19.1 + 4.3 5.0 z 0.7
50.6 + 3.5 45.1 5 5.3 46.5 5 - 3.5
c.001 c.01 c.001
131.6 + 7.1 29.0 + 2.3 3.3 5 0.6
81.4 + 7.8 34.0 T 3.0 13.6 - 2.7
c.001 NS c.01
317.5 + 13.7 17.4 5 - 2.5
c.001 NS
141.6 + 10.4 12.4 - 1.6
The putative substance P neurotoxin, capsaicin, has been reported to deplete the peptide from primary afferent fibers in adult rats (13,141. Parenteral administration of high doses of capsaicin to guinea pigs produced a reduction in substance P levels that was limited to dorsal roots plus ganglia and dorsal spinal cord (Table II). The reduction was 78% in dorsal roots and 43% in spinal cord. There were no significant changes in gastrointestinal tissue or in brain corpus striatum or hypothalamus.
385
TABLE II Effect of parenteral capsaicin administration on substance P levels in guinea pigs. Values are pmole/g wet weight of tissue with mean 2 s.e.m. shown. Capsaicin was administered in consecutive daily doses of 50,200,200,400,400 mg/kg S.C. and animals were killed 8 days after the last injection. Capsaicin (n = 4)
Control (n = 5) Duodenum Ileum Colon Hypothalamus Corpus striatum Dorsal root plus ganglion Dorsal cord Ventral cord
P
93.0 + 18.3 73.9 5 12.6 65.4 E 4.6
112.1 + 11.4 115.8 5 23.4 81.1 z 5.1
NS NS NS
129.9 + 24.3 56.1 + 5.1 24.6 ;: 6.0
149.9 + 32.2 95.3 7 38.1 5.4 5 0.5
NS NS <.025
291.5 + 36.4 17.1 + 3.0
165.6 + 25.1 16.8 i 5.0
c.025 NS
DISCUSSION Our results clearly show that gastrointestinal tissues of guinea pigs contain substantially higher levels of substance P than similar tissues from rats. This is in contrast to early bioassay determinations which suggested that the two species had similar levels in gut tissue (7). The bioassay results, however, are extremely equivocal since early experimenters used impure tissue extracts containing many gastrointestinally active agents (20). Hokfelt et al. (21) have reported no species difference in number of substance P cell bodies in colchicine-treated gut tissues from rats and guinea pigs. However, even though there are definite intrinsic substance P-containing neurons in the gut (22,231, there is also evidence for extrinsic substance P innervation of the gastrointestinal system (23,24). Species differences in this extrinsic innervation, undetectable by counting of peptide cell bodies in gut tissue, could account for the differences in substance P levels we observed between rat and guinea pig intestinal tissues. In support of this possibility is the observation by Hokfelt et al. (25) that mesenteric ganglia of guinea pigs contain more substance P immunofluorescent fibers than ganglia from rats. In the central nervous systems of the two species, there were similarities and differences in substance P levels. While levels were comparable in corpus striatum of rats and guinea pigs, rats had significantly higher levels of the peptide in the hypothalamus. Whether the higher levels in rat hypothalamus result from more substance P-containing neurons intrinsic to the hypothalamus or from more peptide-containing afferents to the hypothalamus remains to be determined. Substance P levels were similar in ventral spinal cord from both species whereas guinea pigs had over P-fold higher peptide levels than rats ir dorsal cord. That the higher levels in guinea pig dorsal cord result from 386
substance P-containing somatic primary afferents is suggested by the 4-fold higher peptide levels in guinea pig dorsal roots compared to rats. This finding indicates either that guinea pigs have a greater number of substance P-containing primary afferent neurons or that the substance P-containing neurons present in guinea pigs contain higher levels of the peptide compared Greater numbers of substance P immunopositive fibers in to those of rats. guinea pig mesenteric ganglia compared to ganglia of rats and the suggestion that some of these originate from neurons in spinal sensory ganglia support the former of these two possibilities (25,26). Capsaicin, the major irritant in hot peppers of the plant genus Capsicum, has recently been reported to produce a depletion of substance P from primary afferents upon parenteral administration to adult rats (13,141. The results of the present study indicate that capsaicin has a similar neurotoxic action in adult guinea pigs. Substance P levels were reduced by the agent extensively in dorsal roots and moderately in dorsal spinal cord. There were no changes in levels of the peptide in the two brain regions studied or in any The reductions in substance P levels in part of the gastrointestinal tract. guinea pig afferents were similar in magnitude to those observed in rats (13; Buck et al., unpublished data). Thus, despite the marked differences in substance P levels in primary afferent neuronal tissues of rats and guinea pigs, peptide-containing afferents in the two species exhibit at least a qualitatively similar susceptibility to the effects of capsaicin. The possibility that there is a difference between the two species in the structure of substance P in the immunoreactive region of the molecule cannot be unequivocally ruled out as the reason for our results. However, non-immunological analytical methods employed by others have not detected species differences in peptide structure. Substantially higher levels of substance P in guinea pig gastrointestinal and primary afferent tissue may indicate a more important neurophysiological role of the peptide in these systems of the guinea pig compared to the rat. The greater sensitivity of guinea pig gut tissue to the contractile effects of substance P supports this contention for guinea pig gastrointestinal function (3,5). Similarly, we have observed that capsaicin treatment of adult guinea pigs results in a profound thermal analgesia, an effect not seen in adult rats treated with capsaicin (14; Buck et al., submitted). In toto, these results suggest that the guinea pig may be a more useful species than the rat in which to investigate the biological roles of substance P and the physiological and pharmacological changes consequent to capsaicin neurotoxicity. ACKNOWLEDGEMENTS I___ Appreciation is extended to Dr. Marvin R. Brown, The Salk Institute, for generous supplies of substance P antiserum. Supported by USPHS grants DA02163, NS15420, and MH30626. H.1. Yamamura is a recipient of an RSDA, Type 11 (EIH-00095), from the National Institute of Mental Health.
387
REFERENCES :-_1. von Euler US, Gaddum JH.
certain tissue extracts.
(1931) An unidentified depressor substance in J. Physiol. (London) 72, 74-87.
2. Gaddum JH, Schild H. (1934) Depressor substances in extracts of intestine. J. Physiol. (London) 83, 1-14. 3. Chang MM, Leeman SE. (1970) Isolation of a sialogogic peptide from bovine hypothalamic tissue and its characterization as substance P. J. Biol. Chem. 245, 4784-4790. 4. Chang MM, Leeman SE, Niall HD. (1971) Amino-acid sequence of substance P. Nature New Biology 232, 86-87. 5. Tregear GW, Niall HD, Potts JT et al. (1971) Nature New Biology 232, 87-89.
Synthesis of substance P.
6. Powell D, Leeman S, Tregear GW et al. (1973) Radioimmunoassay for substance P. Nature New Biology 241, 252-254. 7. Lembeck F, Zetler G. (1962) Substance P: a polypeptide of possible physiological significance, especially within the nervous system. Inter. Rev. Neurobiol. 4, 159-215. a.
Bury RW, Mashford ML. (1977) Substance P: its pharmacology and physiological roles. Aust. J. Exp. Biol. Med. Sci. 55, 671-735.
9.
Nicoll RA, Schenker C, Leeman SE. (1980) Substance P as a transmitter candidate. Ann. Rev. Neurosci. 3, 227-268.
10.
Otsuka M, Takahashi T. (1977) Putative peptide neurotransmitters. Ann. Rev. Pharmacol. 17, 425-439.
11.
Bury RW, Mashford ML. (1976) Biological activity of C-terminal partial sequences of substance P. J. Med. Chem. 19, 854-856.
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Accepted
3 March,
1981
389