- Email: [email protected]
Distribution of substance P in the rat gastrointestinal tract — Lack of effect of capsaicin pretreatment
European Journal o f Pharmacology, 61 (1980) 303--307
303
© Elsevier/North-Holland Biomedical Press
Short communication DISTRIBUTION OF SUBSTANCE P IN THE RAT GASTROINTESTINAL TRACT -- LACK OF EFFECT OF CAPSAICIN PRETREATMENT PETER HOLZER, R A I N E R GAMSE and F R E D LEMBECK
lnstitut fiir Experimentelle und Klinische Pharmakoiogie der Universitd't Graz, Universitd~splatz 4, A-8010 Graz, Austria Received 4 December 1979, accepted 5 December 1979
P. HOLZER, R. GAMSE and F. LEMBECK, Distribution o f substance P in the rat gastrointestinal tract -- lack o f effect ofcapsaicin pretreatment, European J. Pharmacol. 61 (1980) 303--307. A new m e t h o d for extraction of immunoreactive substance P (I-SP) from rat intestine including pulverization of tissue frozen in liquid nitrogen and extraction with acid acetone is described. Using this method, amounts of I-SP in the rat intestine were found to be higher than previously reported. The highest concentrations of I-SP were found in the small intestine. Capsaicin pretreatment of newborn or adult rats had no effect on intestinal I-SP concentrations indicating that intrinsic SP neurones are capsaicin-insensitive. Substance P
Rat gastrointestinal tract
Capsaicin
1. Introduction Substance P (SP) has been demonstrated in the gastrointestinal tract by bioassay (Pernow, 1953) and radioimmunoassay (Nilsson and Brodin, 1977; Gilbert and Emson, 1979). Immunohistochemical studies have revealed that SP is located within neuronal and endocrine cells in the gut (HSkfelt et al., 1977). SP-containing neurones in the gut are predominantly intrinsic (Franco et al., 1979), with only a small number of extrinsic fibres (HSkfelt et al., 1977; Gamse et al., 1979a). Neonatal capsaicin treatment has been shown to cause degeneration of those sensory neurones which are chemosensitive (Jancs5 et al., 1977) and an irreversible decrease of SP in regions containing primary sensory neurones (Gamse et al., 1979b). In the present study we have investigated the distribution of SP within the gastrointestinal tract using an extraction procedure that results in higher SP values than previously reported (Pernow, 1953; Nilsson and Brodin, 1977). We have
also examined the effect of neonatal capsaicin treatment on the SP content of the gut.
2. Materials and methods Sprague-Dawley rats (210--280 g) of either sex, fasted overnight, were killed by a blow on the neck. The following parts of the gastrointestinal tract were rapidly removed, blotted and frozen on dry ice: oesophagus, fundus, antrum, duodenum, proximal jejunum, distal ileum, proximal colon, and, in addition, the submandibular salivary gland. The tissue was weighed and extracted on the same day. Method 1 : Tissue was added to 10 volumes of boiling 0.01 N HC1 according to Nilsson and Brodin (1977). After boiling for 10 min, the .samples were centrifuged and the supernatant was lyophilized. Method 2: Tissue was frozen in liquid nitrogen and pulverized with a Braun®-Melsungen dismembrator within 30 sec. To this were added 8 volumes of ice-cold
304
acetone-1 N HC1 (100 : 3, v/v, according to Chang and Leeman, 1970) and the tissue was extracted by shaking for another 30 sec. The samples were centrifuged, the pellets washed with 3 ml acetone-0.01 N HC1 (80 : 20, v/v) and the supernatants were combined. Lipids were removed b y washing with 2 × 3 ml petroleum ether (bp 40--60°C). Acetone was evaporated at 60°C and the aqueous phase lyophilized. Samples were dissolved in assay buffer and immunoreactive substance P (I-SP) was measured by radioimmunoassay (Gamse et al., 1979a). Samples were assayed in duplicate at 3 dilutions. Newborn rats were injected with a single dose of 50 m g . kg -~ s.c. capsaicin (Merck, Darmstadt) on the second day of life. Control rats received solvent only (10% ethanol, 10% Tween 80 in saline). Adult rats were treated with capsaicin on 4 consecutive days: On the first and second day they received 50 mg • kg-~ s.c. capsaicin. On the third day they were injected with 20 mg • kg -~ i.p. capsaicin, 20 min after they had received 5 mg • kg -~ i.p. isoprenaline, 5 mg • kg -1 i.p. mepyramine, and 10 m g . kg -~ i.p. atropine. Without this pretreatment, rats did n o t survive the i.p. injection of 20 mg • kg -~ capsaicin. On the fourth day the animals received 30 mg • kg -1 i.p. capsaicin, 20 min after pretreatment with isoprenaline, mepyramine, and atropine. Control rats were pretreated in the same way b u t received solvent instead of capsaicin solution.
3. Results
3.1. Comparison o f the two extraction methods The slope of the ileum samples extracted with acid acetone was parallel to the standard curve. Samples extracted with dilute HC1 exhibited a flatter slope. The I-SP content of the samples was calculated from the amount of I-SP displacing 50% of the tracer. Extraction with acid acetone yielded a b o u t 6 times
P. H O L Z E R E T AL.
more I-SP than boiling with dilute HCl: 64.8 + 8.8 ng • g-1 versus 10.2 -+ 1.1 ng • g-1 (n = 6). Recovery of synthetic SP was 86-+ 3% ( n = 4 ) with acid acetone and 8 9 + 4 % (n = 3) with HC1.
3.2. Chromatography o f I-SP extracted from ileum I-SP extracted by the 2 methods was chromatographed on Sephadex G-25 and compared with synthetic SP (fig. 1). Chromatography of an acid acetone extract showed a major peak of immunoreactivity at the position of synthetic SP. This peak contained 98% of the eluted immunoreactivity whereas only 85% of the immunoreactivity of HC1 extracts eluted at the position of synthetic SP. The HC1 extracts contained a b o u t 10% immunoreactivity of smaller molecular weight than SP. Some immunoreactivity running in the void volume was found in b o t h extracts (HC1 extract 5%; acid acetone extract 2%). Total recovery of the applied immunoreactivity was 72% for synthetic SP, 82% for HC1 extracts and 91% for the acid acetone extracts.
3.3. Distribution o f I-SP in the gastrointestinal tract The acid acetone extraction method was used to study the distribution of I-SP in the rat gastrointestinal tract. The highest concentrations were found in the small intestine, the lowest in the antrum and in the oesophagus (table 1). The submandibular gland also contained measurable amounts of I-SP.
3.4. Effect o f capsaicin treatment Two months after capsaicin treatment on the second day of life, acid acetone-extracted I-SP was determined in various parts of the rat gastrointestinal tract. No change in the I-SP content was found in any of the parts investigated (table 1). For comparison, the SP content of the dorsal half of the spinal cord was significantly decreased to 115 n g . g-i corn-
SUBSTANCE P IN THE INTESTINE
305
pg I-SPI froction 1000
l
500
_
_
_~'--='=~i~_ I
20
~_A'_., / I
30
-A.,,.._ I
40
I
=x~l
50 60 fraction number
Fig. 1. Chromatography on Sephadex G-25. • ...... • synthetic SP, × × acid acetone extract and A A m e a n of 2 HCI extracts from ileum: column 1.2 X 95 cm, eluent: phosphate-buffered saline p H 7.4, fraction size 2 rnl.The immunoreactivity of every fraction was assayed directly. V0 was determined with Blue Dextran.
p a r e d w i t h 2 4 2 n g . g-1 in t h e c o n t r o l rats, and t h e SP c o n t e n t o f t h e vagus nerve was r e d u c e d t o 19.6 n g . g-i c o m p a r e d with 51.0 n g . g-~ in the c o n t r o l s ( G a m s e e t al., 1 9 7 9 b ) . Also capsaicin p r e t r e a t m e n t o f a d u l t rats did n o t a f f e c t t h e I-SP c o n t e n t o f t h e gut as determ i n e d in t h e d u o d e n u m a n d distal ileum. I-SP was e x t r a c t e d w i t h acid a c e t o n e 4 d a y s a f t e r
t h e last i.p. injection o f capsaicin. In t h e cont r o l rats, t h e I-SP c o n t e n t o f t h e d u o d e n u m was 6 4 . 3 + 3.4 n g • g-~ and t h a t o f t h e distal ileum was 68.7 +- 3.6 ng • g-l; in t h e capsaicinp r e t r e a t e d animals the d u o d e n u m h a d an I-SP c o n t e n t o f 63.8 + 2.1 ng • g-~ and t h e distal ileum a c o n t e n t o f 6 9 . 6 -+ 4.3 n g . g-~ (n = 5).
4. Discussion
TABLE 1 Distribution of I-SP in the rat gastrointestinal tract
and effect of neonatal capsaicin pretreatment. Rats were injected with 50 mg• kg -! s.c. capsaicin or control solution on the second day of life; 2 months later, I-SP was determined after acid acetone extraction. Values are given in ng" g-I and represent the mean _+S.E.M. from 6 rats. Control Oesophagus
1.4 _+0.2
Fundus
24.9 + 1.6
Antrum
9.2 57.6 62.4 64.5 27.1 4.1
Duodenum
Proximal jejunum Distal ileum Proximal colon Submandibular gland
_+0.6 _+2.8 + 6.4 _+6.2 _+2.8 -+ 0.5
Capsaicin 1.0 -+ 0.2 28.9 -+ 3.9 10.8 + 1.8 56.4 -+ 4.4 64.4 -+ 5.2 70.8_+ 4.8 26.0 + 1.1 4.4 -+ 0.2
T h e results s h o w t h a t acid a c e t o n e extract i o n o f SP f r o m gut tissue yields m u c h higher values t h a n HC1 e x t r a c t i o n , whereas t h e r e c o v e r y o f s y n t h e t i c SP w i t h o u t tissue is a b o u t the same. It can, t h e r e f o r e , be assumed t h a t it is t h e d i f f e r e n t e x t r a c t i o n f r o m the tissue t h a t i m p r o v e s the efficiency. O u r m e t h o d includes p u l v e r i z a t i o n o f t h e t i s s u e f r o z e n in liquid n i t r o g e n , whereas, in t h e o t h e r m e t h o d , t h e tissue is o n l y boiled w i t h o u t h o m o g e n i s a tion. T h e r e f o r e , o u r m e t h o d is p r o b a b l y m o r e e f f i c i e n t in e x t r a c t i n g S P f r o m cellular stores and also in inhibiting e n z y m a t i c d e g r a d a t i o n , especially b y t h e m u c o s a l layer, since t h e tissue is f r o z e n all t h e t i m e until t h e a d d i t i o n o f acid a c e t o n e . C h r o m a t o g r a p h y o n S e p h a d e x G - 2 5 o f an acid a c e t o n e e x t r a c t f r o m t h e
306
ileum revealed that 98% of the immunoreactivity behaved like synthetic SP, whereas one major and two minor peaks of immunoreactivity were seen in chromatograms of HC1 extracts. The presence of cross-reacting substances in these extracts presumably explains the non-identity of the slopes of sample dilutions with the SP standard curve. Three major peaks of immunoreactivity were found by Nilsson and Brodin (1977), when HC1 extracts were chromatographed on Sephadex G-50. The finding of much lower amounts of crossreacting substances in our chromatograms may be due to differences in the specificity of the antibodies used. There was and uneven distribution of SP in the rat gastrointestinal tract. All parts of the small intestine contained a b o u t twice the concentration found in any other part of the gut. This differs markedly from the distribution reported by Nilsson and Brodin (1977), where the highest SP values occurred in the colon, the jejunum, and in the fundus. There is, however, agreement that the fundus contains much .more SP than the antrum and that the lowest values are found in the oesophagus. The SP content of the d u o d e n u m found with acid acetone extraction is very similar to that obtained b y extracting with boiling 1 M acetic acid (Gilbert and Emson, 1979). The presence of SP in the salivary gland confirms the finding of HSkfelt et al. (1977) of SP-positive nerve fibres in this organ. The main sources of SP in the gut are the endocrine cells (HSkfelt et al., 1977) and intrinsic neurones, since extrinsic denervation does n o t decrease the gastrointestinal SP content (Franco et al., 1979). Additionally, some SP neurones, running in the vagus nerve (Gamse et al., 1979a) and sympathetic nerves (HSkfelt et al., 1977) t e r m i n a t e in the gut. Neonatal capsaicin pretreatment caused degeneration of SP neurones in the vagus nerve (Gamse et ai., 1979b), did n o t change the SP content of the gut. Thus it seems that the SP neurones of the vagus do n o t significantly contribute to the intestinal Sp content and that the intrinsic SP neurones are capsaicin-insensitive. However, reduction
P. HOLZER ET AL.
of the SP content of extrinsic neurones may have gone undetected, since SP was only measured in sections of the whole gut. The fact that the intestinal SP content was n o t changed 2 months after neonatal capsaicin pretreatment may have been due to an early reversible depletion of SP in the gut or to the inadequacy of s.c. administration. To clarify this point, we also treated adult rats with capsaicin, first s.c. and then i.p. Capsaicin pretreatment of adult rats is known to decrease the SP content of rat skin (Gamse et al., 1979b) and of the dorsal half of the spinal cord (Jessell et al., 1978). There was, however, no change of the SP content in the 2 intestinal segments investigated, the duodenum and the distal ileum, as determined 4 days after the last i.p. injection of capsaicin. Thus capsaicin is n o t likely to affect intrinsic SP neurones of the gut. This further confirms the suggestion that -- in so far as SP neurones are concerned -- capsaicin acts selectively on primary afferent SP neurones (Gamse et al., 1979b).
Acknowledgements This work was supported by grant No. 3506 of the Austrian Scientific Research Funds. SP antibody was a generous gift of Dr. S.E. Leeman. We would like to thank Mrs. U. Hetzendorf and Mr. W. Schluet for their technical assistance.
References Chang, M.M. and S.E. Leeman, 1970, Isolation of a sialogogic peptide from bovine hypothalamic tissue and its characterization as substance P, J. Biol. Chem. 245, 4784. Franco, R., M. Costa and J.B. Furness, 1979, Evidence that axons containing substance P in the guinea-pig ileum are of intrinsic origin, NaunynSchmiedeb. Arch. Pharmacol. 307, 57. Gamse, R., F. Lembeck and A.C. Cuello, 1979a, Substance P in the vagus nerve: Immunochemical and immunohistochemical evidence for axoplasmic transport, Naunyn-Schmiedeb. ArCh. Pharmacol. 306, 37. Gamse, R., P. Holzer and F. Lembeck, 1979b, Decrease o f substance P in primary afferent neu-
SUBSTANCE
P IN T H E I N T E S T I N E
rones and impairment of neurogenic plasma extravasation by capseicin, Br. J. Pharmacol. (in press). Gilbert, R.F.T. and P.C. Emson, 1979, Substance P in rat CNS and duodenum during development, Brain Res. 171,166. H~kfelt, T., O. Johansson, J.-O. Kellerth, A. Ljungdahl, G. Nilsson, A. Nygards and B. Pernow, 1977, Immunohistochemical distribution of substance P, in: Substance P, eds. U.S. Von Euler and B. Pern o w (Raven Press, N e w York) p. 117. Jancs6, G., E. Kiraly and A. JancsS-G~bor, 1977, Pharmacologically induced selective degeneration of chemosensitive primary sensory neurones, Nature 270,741.
307 Jessell, T.M., L.L. Iversen and A.C. Cuello, 1978, Capsaicin-induced depletion of substance P from primary sensory neurones, Brain Res. 152, 183. Nilsson, G. and E. Brodin, 1977, Tissue distribution of substance P-like immunoreactivity in dog, cat, rat, and mouse, in: Substance P, eds. U.S. Von Euler and B. Pernow (Raven Press, N e w York) p. 49. Pernow, B., 1953, Studies on substance P: Purification, occurrence, and biological actions, Acta Physiol. Scand. 29, Suppl. 105, 1.
303
© Elsevier/North-Holland Biomedical Press
Short communication DISTRIBUTION OF SUBSTANCE P IN THE RAT GASTROINTESTINAL TRACT -- LACK OF EFFECT OF CAPSAICIN PRETREATMENT PETER HOLZER, R A I N E R GAMSE and F R E D LEMBECK
lnstitut fiir Experimentelle und Klinische Pharmakoiogie der Universitd't Graz, Universitd~splatz 4, A-8010 Graz, Austria Received 4 December 1979, accepted 5 December 1979
P. HOLZER, R. GAMSE and F. LEMBECK, Distribution o f substance P in the rat gastrointestinal tract -- lack o f effect ofcapsaicin pretreatment, European J. Pharmacol. 61 (1980) 303--307. A new m e t h o d for extraction of immunoreactive substance P (I-SP) from rat intestine including pulverization of tissue frozen in liquid nitrogen and extraction with acid acetone is described. Using this method, amounts of I-SP in the rat intestine were found to be higher than previously reported. The highest concentrations of I-SP were found in the small intestine. Capsaicin pretreatment of newborn or adult rats had no effect on intestinal I-SP concentrations indicating that intrinsic SP neurones are capsaicin-insensitive. Substance P
Rat gastrointestinal tract
Capsaicin
1. Introduction Substance P (SP) has been demonstrated in the gastrointestinal tract by bioassay (Pernow, 1953) and radioimmunoassay (Nilsson and Brodin, 1977; Gilbert and Emson, 1979). Immunohistochemical studies have revealed that SP is located within neuronal and endocrine cells in the gut (HSkfelt et al., 1977). SP-containing neurones in the gut are predominantly intrinsic (Franco et al., 1979), with only a small number of extrinsic fibres (HSkfelt et al., 1977; Gamse et al., 1979a). Neonatal capsaicin treatment has been shown to cause degeneration of those sensory neurones which are chemosensitive (Jancs5 et al., 1977) and an irreversible decrease of SP in regions containing primary sensory neurones (Gamse et al., 1979b). In the present study we have investigated the distribution of SP within the gastrointestinal tract using an extraction procedure that results in higher SP values than previously reported (Pernow, 1953; Nilsson and Brodin, 1977). We have
also examined the effect of neonatal capsaicin treatment on the SP content of the gut.
2. Materials and methods Sprague-Dawley rats (210--280 g) of either sex, fasted overnight, were killed by a blow on the neck. The following parts of the gastrointestinal tract were rapidly removed, blotted and frozen on dry ice: oesophagus, fundus, antrum, duodenum, proximal jejunum, distal ileum, proximal colon, and, in addition, the submandibular salivary gland. The tissue was weighed and extracted on the same day. Method 1 : Tissue was added to 10 volumes of boiling 0.01 N HC1 according to Nilsson and Brodin (1977). After boiling for 10 min, the .samples were centrifuged and the supernatant was lyophilized. Method 2: Tissue was frozen in liquid nitrogen and pulverized with a Braun®-Melsungen dismembrator within 30 sec. To this were added 8 volumes of ice-cold
304
acetone-1 N HC1 (100 : 3, v/v, according to Chang and Leeman, 1970) and the tissue was extracted by shaking for another 30 sec. The samples were centrifuged, the pellets washed with 3 ml acetone-0.01 N HC1 (80 : 20, v/v) and the supernatants were combined. Lipids were removed b y washing with 2 × 3 ml petroleum ether (bp 40--60°C). Acetone was evaporated at 60°C and the aqueous phase lyophilized. Samples were dissolved in assay buffer and immunoreactive substance P (I-SP) was measured by radioimmunoassay (Gamse et al., 1979a). Samples were assayed in duplicate at 3 dilutions. Newborn rats were injected with a single dose of 50 m g . kg -~ s.c. capsaicin (Merck, Darmstadt) on the second day of life. Control rats received solvent only (10% ethanol, 10% Tween 80 in saline). Adult rats were treated with capsaicin on 4 consecutive days: On the first and second day they received 50 mg • kg-~ s.c. capsaicin. On the third day they were injected with 20 mg • kg -~ i.p. capsaicin, 20 min after they had received 5 mg • kg -~ i.p. isoprenaline, 5 mg • kg -1 i.p. mepyramine, and 10 m g . kg -~ i.p. atropine. Without this pretreatment, rats did n o t survive the i.p. injection of 20 mg • kg -~ capsaicin. On the fourth day the animals received 30 mg • kg -1 i.p. capsaicin, 20 min after pretreatment with isoprenaline, mepyramine, and atropine. Control rats were pretreated in the same way b u t received solvent instead of capsaicin solution.
3. Results
3.1. Comparison o f the two extraction methods The slope of the ileum samples extracted with acid acetone was parallel to the standard curve. Samples extracted with dilute HC1 exhibited a flatter slope. The I-SP content of the samples was calculated from the amount of I-SP displacing 50% of the tracer. Extraction with acid acetone yielded a b o u t 6 times
P. H O L Z E R E T AL.
more I-SP than boiling with dilute HCl: 64.8 + 8.8 ng • g-1 versus 10.2 -+ 1.1 ng • g-1 (n = 6). Recovery of synthetic SP was 86-+ 3% ( n = 4 ) with acid acetone and 8 9 + 4 % (n = 3) with HC1.
3.2. Chromatography o f I-SP extracted from ileum I-SP extracted by the 2 methods was chromatographed on Sephadex G-25 and compared with synthetic SP (fig. 1). Chromatography of an acid acetone extract showed a major peak of immunoreactivity at the position of synthetic SP. This peak contained 98% of the eluted immunoreactivity whereas only 85% of the immunoreactivity of HC1 extracts eluted at the position of synthetic SP. The HC1 extracts contained a b o u t 10% immunoreactivity of smaller molecular weight than SP. Some immunoreactivity running in the void volume was found in b o t h extracts (HC1 extract 5%; acid acetone extract 2%). Total recovery of the applied immunoreactivity was 72% for synthetic SP, 82% for HC1 extracts and 91% for the acid acetone extracts.
3.3. Distribution o f I-SP in the gastrointestinal tract The acid acetone extraction method was used to study the distribution of I-SP in the rat gastrointestinal tract. The highest concentrations were found in the small intestine, the lowest in the antrum and in the oesophagus (table 1). The submandibular gland also contained measurable amounts of I-SP.
3.4. Effect o f capsaicin treatment Two months after capsaicin treatment on the second day of life, acid acetone-extracted I-SP was determined in various parts of the rat gastrointestinal tract. No change in the I-SP content was found in any of the parts investigated (table 1). For comparison, the SP content of the dorsal half of the spinal cord was significantly decreased to 115 n g . g-i corn-
SUBSTANCE P IN THE INTESTINE
305
pg I-SPI froction 1000
l
500
_
_
_~'--='=~i~_ I
20
~_A'_., / I
30
-A.,,.._ I
40
I
=x~l
50 60 fraction number
Fig. 1. Chromatography on Sephadex G-25. • ...... • synthetic SP, × × acid acetone extract and A A m e a n of 2 HCI extracts from ileum: column 1.2 X 95 cm, eluent: phosphate-buffered saline p H 7.4, fraction size 2 rnl.The immunoreactivity of every fraction was assayed directly. V0 was determined with Blue Dextran.
p a r e d w i t h 2 4 2 n g . g-1 in t h e c o n t r o l rats, and t h e SP c o n t e n t o f t h e vagus nerve was r e d u c e d t o 19.6 n g . g-i c o m p a r e d with 51.0 n g . g-~ in the c o n t r o l s ( G a m s e e t al., 1 9 7 9 b ) . Also capsaicin p r e t r e a t m e n t o f a d u l t rats did n o t a f f e c t t h e I-SP c o n t e n t o f t h e gut as determ i n e d in t h e d u o d e n u m a n d distal ileum. I-SP was e x t r a c t e d w i t h acid a c e t o n e 4 d a y s a f t e r
t h e last i.p. injection o f capsaicin. In t h e cont r o l rats, t h e I-SP c o n t e n t o f t h e d u o d e n u m was 6 4 . 3 + 3.4 n g • g-~ and t h a t o f t h e distal ileum was 68.7 +- 3.6 ng • g-l; in t h e capsaicinp r e t r e a t e d animals the d u o d e n u m h a d an I-SP c o n t e n t o f 63.8 + 2.1 ng • g-~ and t h e distal ileum a c o n t e n t o f 6 9 . 6 -+ 4.3 n g . g-~ (n = 5).
4. Discussion
TABLE 1 Distribution of I-SP in the rat gastrointestinal tract
and effect of neonatal capsaicin pretreatment. Rats were injected with 50 mg• kg -! s.c. capsaicin or control solution on the second day of life; 2 months later, I-SP was determined after acid acetone extraction. Values are given in ng" g-I and represent the mean _+S.E.M. from 6 rats. Control Oesophagus
1.4 _+0.2
Fundus
24.9 + 1.6
Antrum
9.2 57.6 62.4 64.5 27.1 4.1
Duodenum
Proximal jejunum Distal ileum Proximal colon Submandibular gland
_+0.6 _+2.8 + 6.4 _+6.2 _+2.8 -+ 0.5
Capsaicin 1.0 -+ 0.2 28.9 -+ 3.9 10.8 + 1.8 56.4 -+ 4.4 64.4 -+ 5.2 70.8_+ 4.8 26.0 + 1.1 4.4 -+ 0.2
T h e results s h o w t h a t acid a c e t o n e extract i o n o f SP f r o m gut tissue yields m u c h higher values t h a n HC1 e x t r a c t i o n , whereas t h e r e c o v e r y o f s y n t h e t i c SP w i t h o u t tissue is a b o u t the same. It can, t h e r e f o r e , be assumed t h a t it is t h e d i f f e r e n t e x t r a c t i o n f r o m the tissue t h a t i m p r o v e s the efficiency. O u r m e t h o d includes p u l v e r i z a t i o n o f t h e t i s s u e f r o z e n in liquid n i t r o g e n , whereas, in t h e o t h e r m e t h o d , t h e tissue is o n l y boiled w i t h o u t h o m o g e n i s a tion. T h e r e f o r e , o u r m e t h o d is p r o b a b l y m o r e e f f i c i e n t in e x t r a c t i n g S P f r o m cellular stores and also in inhibiting e n z y m a t i c d e g r a d a t i o n , especially b y t h e m u c o s a l layer, since t h e tissue is f r o z e n all t h e t i m e until t h e a d d i t i o n o f acid a c e t o n e . C h r o m a t o g r a p h y o n S e p h a d e x G - 2 5 o f an acid a c e t o n e e x t r a c t f r o m t h e
306
ileum revealed that 98% of the immunoreactivity behaved like synthetic SP, whereas one major and two minor peaks of immunoreactivity were seen in chromatograms of HC1 extracts. The presence of cross-reacting substances in these extracts presumably explains the non-identity of the slopes of sample dilutions with the SP standard curve. Three major peaks of immunoreactivity were found by Nilsson and Brodin (1977), when HC1 extracts were chromatographed on Sephadex G-50. The finding of much lower amounts of crossreacting substances in our chromatograms may be due to differences in the specificity of the antibodies used. There was and uneven distribution of SP in the rat gastrointestinal tract. All parts of the small intestine contained a b o u t twice the concentration found in any other part of the gut. This differs markedly from the distribution reported by Nilsson and Brodin (1977), where the highest SP values occurred in the colon, the jejunum, and in the fundus. There is, however, agreement that the fundus contains much .more SP than the antrum and that the lowest values are found in the oesophagus. The SP content of the d u o d e n u m found with acid acetone extraction is very similar to that obtained b y extracting with boiling 1 M acetic acid (Gilbert and Emson, 1979). The presence of SP in the salivary gland confirms the finding of HSkfelt et al. (1977) of SP-positive nerve fibres in this organ. The main sources of SP in the gut are the endocrine cells (HSkfelt et al., 1977) and intrinsic neurones, since extrinsic denervation does n o t decrease the gastrointestinal SP content (Franco et al., 1979). Additionally, some SP neurones, running in the vagus nerve (Gamse et al., 1979a) and sympathetic nerves (HSkfelt et al., 1977) t e r m i n a t e in the gut. Neonatal capsaicin pretreatment caused degeneration of SP neurones in the vagus nerve (Gamse et ai., 1979b), did n o t change the SP content of the gut. Thus it seems that the SP neurones of the vagus do n o t significantly contribute to the intestinal Sp content and that the intrinsic SP neurones are capsaicin-insensitive. However, reduction
P. HOLZER ET AL.
of the SP content of extrinsic neurones may have gone undetected, since SP was only measured in sections of the whole gut. The fact that the intestinal SP content was n o t changed 2 months after neonatal capsaicin pretreatment may have been due to an early reversible depletion of SP in the gut or to the inadequacy of s.c. administration. To clarify this point, we also treated adult rats with capsaicin, first s.c. and then i.p. Capsaicin pretreatment of adult rats is known to decrease the SP content of rat skin (Gamse et al., 1979b) and of the dorsal half of the spinal cord (Jessell et al., 1978). There was, however, no change of the SP content in the 2 intestinal segments investigated, the duodenum and the distal ileum, as determined 4 days after the last i.p. injection of capsaicin. Thus capsaicin is n o t likely to affect intrinsic SP neurones of the gut. This further confirms the suggestion that -- in so far as SP neurones are concerned -- capsaicin acts selectively on primary afferent SP neurones (Gamse et al., 1979b).
Acknowledgements This work was supported by grant No. 3506 of the Austrian Scientific Research Funds. SP antibody was a generous gift of Dr. S.E. Leeman. We would like to thank Mrs. U. Hetzendorf and Mr. W. Schluet for their technical assistance.
References Chang, M.M. and S.E. Leeman, 1970, Isolation of a sialogogic peptide from bovine hypothalamic tissue and its characterization as substance P, J. Biol. Chem. 245, 4784. Franco, R., M. Costa and J.B. Furness, 1979, Evidence that axons containing substance P in the guinea-pig ileum are of intrinsic origin, NaunynSchmiedeb. Arch. Pharmacol. 307, 57. Gamse, R., F. Lembeck and A.C. Cuello, 1979a, Substance P in the vagus nerve: Immunochemical and immunohistochemical evidence for axoplasmic transport, Naunyn-Schmiedeb. ArCh. Pharmacol. 306, 37. Gamse, R., P. Holzer and F. Lembeck, 1979b, Decrease o f substance P in primary afferent neu-
SUBSTANCE
P IN T H E I N T E S T I N E
rones and impairment of neurogenic plasma extravasation by capseicin, Br. J. Pharmacol. (in press). Gilbert, R.F.T. and P.C. Emson, 1979, Substance P in rat CNS and duodenum during development, Brain Res. 171,166. H~kfelt, T., O. Johansson, J.-O. Kellerth, A. Ljungdahl, G. Nilsson, A. Nygards and B. Pernow, 1977, Immunohistochemical distribution of substance P, in: Substance P, eds. U.S. Von Euler and B. Pern o w (Raven Press, N e w York) p. 117. Jancs6, G., E. Kiraly and A. JancsS-G~bor, 1977, Pharmacologically induced selective degeneration of chemosensitive primary sensory neurones, Nature 270,741.
307 Jessell, T.M., L.L. Iversen and A.C. Cuello, 1978, Capsaicin-induced depletion of substance P from primary sensory neurones, Brain Res. 152, 183. Nilsson, G. and E. Brodin, 1977, Tissue distribution of substance P-like immunoreactivity in dog, cat, rat, and mouse, in: Substance P, eds. U.S. Von Euler and B. Pernow (Raven Press, N e w York) p. 49. Pernow, B., 1953, Studies on substance P: Purification, occurrence, and biological actions, Acta Physiol. Scand. 29, Suppl. 105, 1.