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The regional distribution of kassinin-like immunoreactivity in central and peripheral tissues of the cat
228
Brain Ife,~earch, 34[ i it~5~ 228--232 F Isexicr
BRE 10933
The Regional Distribution of Kassinin-Like Immunoreactivity in Central and Peripheral Tissues of the Cat JOHN C. HUNTER, PATRICIA A. HANNAH and JOHN E. MAGGIO* MRC Neurochemical Pharmacology Unit, Medical Research Council Centre, Medical School, Cambridge CB2 2QH (U. K. ) (Accepted November 20th, 1984) Key words: substance K - - neuromedin K - - substance P - - regional distribution - - central nervous system - - gastrointestinal tract
The regional distribution of kassinin-like immunoreactivity (KLI) was investigated by radioimmunoassay in central and peripheral tissues of the cat. In the cat brain KLI was found to have a widespread distribution with the highest concentrations present in the substantia nigra followed by the hypothalamus and caudate nucleus. Moderate levels were detected in the spinal cord, brainstem and thalamus but only low levels were found in the frontal cortex and cerebellum. In the periphery KLI was present in moderate amounts in the pituitary but in only low amounts in the dorsal root ganglion. KLI was present throughout the gastrointestinal tract with the highest concentration in the duodenum. The distribution of KLI was found to resemble closely that of substance P-like immunoreactivity (SPLI) although the ratio of SPLI to KLI varied widely throughout the tissues tested. In the frontal cortex and cerebellum the concentration of SPLI was at least 5-fold higher than that of KLI whereas in the pituitary gland, caudate nucleus and terminal ileum the concentrations of SPLI were only 50% higher. High pressure liquid chromatographic analysis of individual tissue extracts was used in order to characterize the KLI. In the caudate nucleus, substantia nigra and hypothalamus the major immunoreactive peak co-eluted with synthetic substance K. In contrast, in the spinal cord and terminal ileum the major peak of KLI co-eluted with synthetic neuromedin K. These results demonstrate that KLI is widely distributed throughout cat tissues and that this distribution closely resembles that of SPLI. However, it also appears that there are important qualitative differences in the KLI between the areas tested.
INTRODUCTION
investigated the regional distribution of kassinin-like
The tachykinin family of peptides share a common, carboxyl-terminus sequence Phe-X-Gty-Leu-
immunoreactivity (KLI) in rat central and peripheral tissues and found it closely resembled the distribution of SP-like immunoreactivity (SPLI) 7.
Met-NH21. In the last year two novel tachykinin peptides with an identical C-terminal sequence to the amphibian tachykinin kassinin (X = Val) ha~e been isolated from bovine and porcine spinal cordS,6, 9. We have recently shown that one of these peptides, substance K (SK), differs from substance P (SP) in its peripheral pharmacology and distribution of binding sites in the central nervous system (CNS)3, ~0. We initially detected substance K in extracts of bovine spinal cord using a C-terminal directed antiserum raised against kassinin9. Using this antiserum we
In the present study we have used the same kassinin antiserum to investigate the regional distribution of KLI in both central and peripheral tissues of the cat. In selected areas we have also analyzed tissue extracts by high-pressure liquid chromatography (HPLC) in order to characterize the relative quantities of kassinin-tike immunoreactive material. We report the existence of marked qualitative differences in the KLI from the respective areas of the cat a n d rat.
* Present address: Neuropsychopharmacology Research Unit, Yale Medical School, 333 Cedar Street, New Haven, CT 06510, U.S.A. Correspondence. J. C. Hunter. Present address: Neuropharmacology Department, Glaxo Group Research Ltd., Ware, Hertfordshire SG 12 ODJ, U.K. 0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
229 MATERIALS AND METHODS Adult male cats (3.0-4.0 kg) were killed by an intraperitoneal injection of pentobarbitone and the tissues rapidly dissected, frozen on dry-ice and weighed. Each tissue sample was then extracted in boiling 10% acetic acid, the homogenate placed in a boiling water bath for 5 min, cooled and then centrifuged at 4000 g for 10 min at room temperature. The supernatant was removed, lyophilized and then resuspended in phosphate assay buffer for measurement of KLI and SPLI by radioimmunoassay. Both KLI and SPLI were measured in every tissue extract. KLI was measured using a rabbit antiserum directed against the carboxyl-terminus of synthetic kassinin. The details of the assay have been previously described 7. The assay shows full cross-reactivity with SK and neuromedin K but negligible cross-reactivity with substance P ( < 0.005). The detection limit of each assay was 10 fmol/assay tube. SPLI was measured using a radioimmunoassay similar to that previously reported from this laboratory 4. Cross-reactivity of kassinin, substance K and neuromedin K in this assay was negligible ( < 0.005). For R P - H P L C analysis, the CNS tissue extracts, prepared as described previously in the text, were reconstituted in 0.1% CF3COOH/20% CH3CN, filtered and injected onto a Ct8 y B o n d a p a k analytical R P - H P L C column equilibrated with the same solvent. The column was eluted at 2 ml/min with a linear gradient of 0.5% CH3CN/min. One millilitre fractions were collected and analyzed for KL1 by R I A . The procedure for the extracts of cat ileum was as described above with the additional step of treatment with 5% trichloracetic acid for 30 min at room temperature to reduce the viscosity of the extract prior to injection onto the R P - H P L C column. KLI in all central and peripheral tissue extracts diluted in parallel to synthetic kassinin, substance K and neuromedin K (X = Val), but not to substance P or tachykinins with other amino acids at position X. RESULTS In the cat brain the highest concentrations of KLI were detected in the substantia nigra with moderate to high amounts found in the hypothalamus and caudate nucleus. The brainstem and thalamus contained
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Fig. 1. RP-HPLC chromatogram of cat spinal cord extract. The upper trace (A) represents SPL] and the lower trace (B) KLI.
An acetic acid extract prepared as described in the next was reconstituted in 0.1% CF)COOH/20% CH3CN,filtered and injected onto a C18 ,uBondapak analytical RP-HPLC column equilibrated with the same solvent. The column was eluted at 2 ml/min with a linear gradient of 0.5% CH3CN/min and 1 ml fractions were collected for radioimmunoassay as in the text. Synthetic standards elute at the following positions in this gradient system: (1) substance K, 11.8 min; (2) substance P, 18.5 min; (3) eledoisin, 20.4 min; (4) neuromedin K, 28.2 min. KLI in the cat spinal cord is chromatographically distinct from all other known tachykinins.
moderate to low concentrations of KL1, the frontal cortex had only very low concentrations and in the cerebellum KLI was only barely detectable (Table I). Using R P - H P L C analysis of the cat brain tissue extracts in order to characterize the KLI, it was found that most of the immunoreactive material eluted at the position of either synthetic substance K or neuromedin K (Figs. 1-3), and was chromatographically distinct from substance P and all other known tachykinins (Fig. 1). In the spinal cord (Fig. 1B), the major immunoreactive peak co-eluted with neuromedin K whereas in the caudate nucleus (Fig. 2A), substantia nigra (Fig. 2B) and hypothalamus (Fig. 3A) the major immunoreactive peak co-eluted with substance K. Furthermore, in the caudate nucleus (Fig. 2A) the amount of immunoreactive material
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Fig. 2. RP-HPLC chromatogram of (A) cat caudate nucleus and (B) cat substantia nigra. The column was eluted under the same conditions as described in Fig. 1. In addition to the synthetic standards, the oxidized forms of the peptides eluted at the following positions in this gradient system: substance K sulphoxide 5.5 rain; neuromedin K monosulphoxide 23.5 rain; neuromedin K disulphoxide 14.5 min. The major peak of KLI co-elutes with synthetic substance K in both the caudate nucleus and substantia nigra. A similar result was obtained in three separate experiments.
Fig. 3. RP-HPLC chromatogram of (A) cat hypothalamusand (B) cat terminal ileum. The column was eluted under the same conditions as described in Fig. 1. The synthetic standards and their sulphoxides eluted at the positions detailed in both Fig. 1 and Fig. 2. The major peak of KLI in the cat hypothalamus coelutes with substance K whereas in the cat ileum the major immunoreactive peak co-elutes with neuromedin K. A similar result was obtained in three separate experiments.
co-eluting with n e u r o m e d i n K was negligible. In p e r i p h e r a l tissues of the cat, K L I was d e t e c t e d in m o d e r a t e amounts in the pituitary but in only low concentrations in the dorsal r o o t ganglia. K L I was present t h r o u g h o u t the gastrointestinal tract with the highest concentration found in the d u o d e n u m (Table II). On R P - H P L C analysis of the cat ileum extract, the m a j o r i m m u n o r e a c t i v e p e a k co-eluted with synthetic n e u r o m e d i n K with only a negligible a m o u n t of K L I co-eluting with substance K (Fig. 3B). The distribution of K L I closely r e s e m b l e d that of substance P-like immunoreactivity (SPLI). However, although the ratio of SPLI to K L I varied in the tissues tested this was not found to be statistically significant (Tables I and II). The concentration of SPLI was at least 6-fold higher than that of K L I in the frontal cortex and c e r e b e l l u m , 4-fold higher in the dorsal root ganglion and b e t w e e n 1.5- and 3-fold higher in a
variety of o t h e r tissues including the hypothalamus, substantia nigra, brainstem, and t h r o u g h o u t the gas~ trointestinal tract. DISCUSSION In the cat brain, the regional distribution of KLI was similar to that found in the raft with the highest concentrations of K L I found in the substantia nigra followed by the h y p o t h a l a m u s and caudate nucleus. Similarly, only low amounts were d e t e c t e d in the cat frontal cortex and cerebellum. The concentrations of K L I in each area of the cat brain was if anything slightly lower than the corresponding values for the rat, although in the cat substantia nigra it was slightly higher. In p e r i p h e r a l cat tissues the concentrations of K L I were similar to those found in the raft. However, the distribution of K L I throughout the gastroin-
231 TABLE I
the concentrations of SPLI were usually between 2
Concentrations of kassinin-like (KL1) and substance P-like (SPL1) immunoreactivity in central tissues of the cat
and 3 times higher than those of KLI. In rat tissues, the concentration of SPLI was consistently twice that of KLI with only minor variation 7, Although tissue
Each value represents the mean _+ S.E.M. of between 4 and 9 observations. Region
KLI
SPL1
SPLI/KLI
(pmol/g wet weight)
Hypothalamus Thalamus Substantia nigra Caudate nucleus Olfactory bulb Frontal cortex Cerebellum Pons/Medulla Spinal cord
172 + 32 49 + 7 635 + 92 110 + 7 20 + 2 7 _+2 2_+0.3 52 _.9_5 65 -+ 9
331 _+ 19 135 _+ 16 1389 _+85 140 _+ 18 28 _+2 35 _+ 12 11_+2 130 + 25 126 _+28
concentrations of SPLI in the cat gastrointestinal tract were similar to previously published data 2, we have been unable to find a similar study on the detailed distribution of SPLI in the cat brain. Perhaps
2.2 _+0.4 2.8 _+0.1 1.8 _+0.2 1.5 _+0.3 1.5 _+0.2 6.3 _+2.5 6.4_+2.8 2.8 _+0.4 2.1 _+0.2
testinal tract was different from the rat with the cat d u o d e n u m and descending colon containing the highest concentrations of KLI. These tissues were found to have the lowest concentrations of KLI in the rat 7. The general distribution of KLI in the cat central and peripheral tissues, as in the cat, closely resembled the distribution of SLPI though a further a n o m a l y was in the ratio of SPLI to KLI. Thus, in certain areas of the cat brain the concentration of SPLI was at least 6
the most surprising differences between the cat and rat were found in the H P L C analysis of the various tissue extracts. In the rat we found that the kassininlike immunoreactive material co-eluting with substance K was consistently the major source of KLI in all tissues tested< In contrast, in cat tissue extracts, although the major source of KLI co-eluted with substance K in the caudate nucleus, substantia nigra and hypothalamus, the reverse situation was found in the spinal cord and ileum where the major peak of immunoreactivity co-eluted with n e u r o m e d i n K. Recently, the m R N A encoding the tachykinin precursor was isolated from bovine striatum 11. Surprisingly, although the sequences of substance P and substance K were present, that of n e u r o m e d i n K was missing. Nevertheless, the presence of these peptides at a genetic level raises the possibility that the differences
times that of KLI whilst in the gastrointestinal tract
observed between the relative a m o u n t s of substance K and n e u r o m e d i n K in the cat and rat spinal cord and ileum could well be due to gene expression.
TABLE II
Furthermore, it should also prove interesting to observe whether this inter-species variation in the ratio of substance K to n e u r o m e d i n K is found in their
Concentrations of kassinin-like (KL1) and substance P-like (SPL D immunoreactivity in peripheral tissues of the cat
Each value represents the mean _+ S.E.M. of between 3 and 5 observations; (n.d. = not determined). Region
KLI
SPL1
SPL1/KL1
Pituitary gland Superior cervical ganglion Dorsal root ganglion Adrenal gland Oesophagus Fundus Antrum Duodenum Jejunum Ileum Colon Pancreas
20.3 + 6.1
24.3 _+7.2
1.2 _+0.1
14.1 _+ 1.3 6.7 _+2.4 < 1 11,3 + 1.1 3.5 _+3.0 6.9 + 1.4 17.4 _+2.0 6.2 _+1.5 6.4 _+2.6 11.0_ + 1.2 1.5 _+0.2
n.d. 26.1 + 8.0 n.d. 31.0 + 4.7 7.6 _+0.9 16.3 _+0.9 40.1 _+8.8 11.8 _+0.4 9.9 _4=-3.2 32.1 _+8.2 n.d.
4.3 _+2.4 2.9 + 0.5 2.2 _+0.9 2.1 + 0.4 2.6 + 0.9 2.1 _+0.4 2.2 _+0.9 2.9+0.8
(pmol/g wet weight)
pharmacology with the possible existence of receptor subtypes. In conclusion, the present study shows that KLI is widely distributed throughout cat central and peripheral tissues where it closely resembles the distribution of SPLI. However, there exist important qualitative differences in the KLI found not only between certain areas of the cat but also between the cat and rat. ACKNOWLEDGEMENTS We thank Mrs. M. W y n n for typing the m a n u script. J.C.H. is a Medical Research Council Postdoctoral Fellow. This work was carried out during the tenure of a Postdoctoral Fellowship to J.E,M. from the Muscular Dystrophy Association.
232 REFERENCES 1 Erspamer, V., The tachykinin peptide family, Trends Neurosci., 4 (1981) 267-269. 2 Holzer, P., Bucsics, A. S. and Lembeck, F., A study of the concentrations of substance P and neurotensin in the gastrointestinal tract of various mammals, Neuroscience. 7 (1982) 2919-2924. 3 Hunter, J. C. and Maggio, J. E., Pharmacological characterization of a novel tachykinin isolated from mammalian spinal cord, Europ. J. Pharmacol., 97 (1984) 159-160. 4 Kanazawa, I. and Jessell, T. M., Post mortem changes and regional distribution of substance P in the rat and mouse nervous system, Brain Research, 117 (1976) 362-367. 5 Kangawa, K., Minamino, N., Fukuda, A. and Matsuo, H., Neuromedin K: a novel mammalian tachykinin identified in porcine spinal cord, Biochem. Biophys. Res. Commun., 114 (1983) 533-540. 6 Kimura, S., Okada, M., Sugita, Y., Kanazawa, I. and Munekata, E., Novel neuropeptides, neurokinin a and 13, isolated from porcine spinal cord, Proe. Jpn. Acad. Ser. B., 59 (1983) 101-104.
7 Maggio, J. E. and Hunter, ,I. (7., Regional distribution ol kassinin-like immunoreactivity in rat central and peripheral tissues and the effect of capsaicin, Brain Research, in press 8 Maggio, J. E. and Hunter, J. C., Chromatographic characterization of kassinin-like immunoreactivity in central and peripheral tissues of the rat, Brain Research, submitted tor publication. 9 Maggio, J. E., Sandberg, B. E. B., Bradley, C. V., lversen, L. L., Santikarn, S., Williams, D. H., Hunter, J. C, and Hanley, M. R., Substance K: a novel tachykinin in mammalian spinal cord. In P. Skrabanek and D. Powell (Eds.), Substance P, Boole Press, Dublin, 1983, pp. 20-21. 10 Mantyh, P. W., Maggio, J. E. and Hunt, S. P , The autoradiographic distribution of kassinin and substance K binding sites is different from that of substance P binding sites, Eutop. J. Pharmacol., in press. 11 Nawa, H., Hirose, T., Takashima, H., inayama, S. and Nakanishi, S., Nucleotide sequences of cloned cDNA for two types of bovine brain substance P precursor, Nature (Lond.), 3(16 (1983) 32-36.
Brain Ife,~earch, 34[ i it~5~ 228--232 F Isexicr
BRE 10933
The Regional Distribution of Kassinin-Like Immunoreactivity in Central and Peripheral Tissues of the Cat JOHN C. HUNTER, PATRICIA A. HANNAH and JOHN E. MAGGIO* MRC Neurochemical Pharmacology Unit, Medical Research Council Centre, Medical School, Cambridge CB2 2QH (U. K. ) (Accepted November 20th, 1984) Key words: substance K - - neuromedin K - - substance P - - regional distribution - - central nervous system - - gastrointestinal tract
The regional distribution of kassinin-like immunoreactivity (KLI) was investigated by radioimmunoassay in central and peripheral tissues of the cat. In the cat brain KLI was found to have a widespread distribution with the highest concentrations present in the substantia nigra followed by the hypothalamus and caudate nucleus. Moderate levels were detected in the spinal cord, brainstem and thalamus but only low levels were found in the frontal cortex and cerebellum. In the periphery KLI was present in moderate amounts in the pituitary but in only low amounts in the dorsal root ganglion. KLI was present throughout the gastrointestinal tract with the highest concentration in the duodenum. The distribution of KLI was found to resemble closely that of substance P-like immunoreactivity (SPLI) although the ratio of SPLI to KLI varied widely throughout the tissues tested. In the frontal cortex and cerebellum the concentration of SPLI was at least 5-fold higher than that of KLI whereas in the pituitary gland, caudate nucleus and terminal ileum the concentrations of SPLI were only 50% higher. High pressure liquid chromatographic analysis of individual tissue extracts was used in order to characterize the KLI. In the caudate nucleus, substantia nigra and hypothalamus the major immunoreactive peak co-eluted with synthetic substance K. In contrast, in the spinal cord and terminal ileum the major peak of KLI co-eluted with synthetic neuromedin K. These results demonstrate that KLI is widely distributed throughout cat tissues and that this distribution closely resembles that of SPLI. However, it also appears that there are important qualitative differences in the KLI between the areas tested.
INTRODUCTION
investigated the regional distribution of kassinin-like
The tachykinin family of peptides share a common, carboxyl-terminus sequence Phe-X-Gty-Leu-
immunoreactivity (KLI) in rat central and peripheral tissues and found it closely resembled the distribution of SP-like immunoreactivity (SPLI) 7.
Met-NH21. In the last year two novel tachykinin peptides with an identical C-terminal sequence to the amphibian tachykinin kassinin (X = Val) ha~e been isolated from bovine and porcine spinal cordS,6, 9. We have recently shown that one of these peptides, substance K (SK), differs from substance P (SP) in its peripheral pharmacology and distribution of binding sites in the central nervous system (CNS)3, ~0. We initially detected substance K in extracts of bovine spinal cord using a C-terminal directed antiserum raised against kassinin9. Using this antiserum we
In the present study we have used the same kassinin antiserum to investigate the regional distribution of KLI in both central and peripheral tissues of the cat. In selected areas we have also analyzed tissue extracts by high-pressure liquid chromatography (HPLC) in order to characterize the relative quantities of kassinin-tike immunoreactive material. We report the existence of marked qualitative differences in the KLI from the respective areas of the cat a n d rat.
* Present address: Neuropsychopharmacology Research Unit, Yale Medical School, 333 Cedar Street, New Haven, CT 06510, U.S.A. Correspondence. J. C. Hunter. Present address: Neuropharmacology Department, Glaxo Group Research Ltd., Ware, Hertfordshire SG 12 ODJ, U.K. 0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
229 MATERIALS AND METHODS Adult male cats (3.0-4.0 kg) were killed by an intraperitoneal injection of pentobarbitone and the tissues rapidly dissected, frozen on dry-ice and weighed. Each tissue sample was then extracted in boiling 10% acetic acid, the homogenate placed in a boiling water bath for 5 min, cooled and then centrifuged at 4000 g for 10 min at room temperature. The supernatant was removed, lyophilized and then resuspended in phosphate assay buffer for measurement of KLI and SPLI by radioimmunoassay. Both KLI and SPLI were measured in every tissue extract. KLI was measured using a rabbit antiserum directed against the carboxyl-terminus of synthetic kassinin. The details of the assay have been previously described 7. The assay shows full cross-reactivity with SK and neuromedin K but negligible cross-reactivity with substance P ( < 0.005). The detection limit of each assay was 10 fmol/assay tube. SPLI was measured using a radioimmunoassay similar to that previously reported from this laboratory 4. Cross-reactivity of kassinin, substance K and neuromedin K in this assay was negligible ( < 0.005). For R P - H P L C analysis, the CNS tissue extracts, prepared as described previously in the text, were reconstituted in 0.1% CF3COOH/20% CH3CN, filtered and injected onto a Ct8 y B o n d a p a k analytical R P - H P L C column equilibrated with the same solvent. The column was eluted at 2 ml/min with a linear gradient of 0.5% CH3CN/min. One millilitre fractions were collected and analyzed for KL1 by R I A . The procedure for the extracts of cat ileum was as described above with the additional step of treatment with 5% trichloracetic acid for 30 min at room temperature to reduce the viscosity of the extract prior to injection onto the R P - H P L C column. KLI in all central and peripheral tissue extracts diluted in parallel to synthetic kassinin, substance K and neuromedin K (X = Val), but not to substance P or tachykinins with other amino acids at position X. RESULTS In the cat brain the highest concentrations of KLI were detected in the substantia nigra with moderate to high amounts found in the hypothalamus and caudate nucleus. The brainstem and thalamus contained
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Fig. 1. RP-HPLC chromatogram of cat spinal cord extract. The upper trace (A) represents SPL] and the lower trace (B) KLI.
An acetic acid extract prepared as described in the next was reconstituted in 0.1% CF)COOH/20% CH3CN,filtered and injected onto a C18 ,uBondapak analytical RP-HPLC column equilibrated with the same solvent. The column was eluted at 2 ml/min with a linear gradient of 0.5% CH3CN/min and 1 ml fractions were collected for radioimmunoassay as in the text. Synthetic standards elute at the following positions in this gradient system: (1) substance K, 11.8 min; (2) substance P, 18.5 min; (3) eledoisin, 20.4 min; (4) neuromedin K, 28.2 min. KLI in the cat spinal cord is chromatographically distinct from all other known tachykinins.
moderate to low concentrations of KL1, the frontal cortex had only very low concentrations and in the cerebellum KLI was only barely detectable (Table I). Using R P - H P L C analysis of the cat brain tissue extracts in order to characterize the KLI, it was found that most of the immunoreactive material eluted at the position of either synthetic substance K or neuromedin K (Figs. 1-3), and was chromatographically distinct from substance P and all other known tachykinins (Fig. 1). In the spinal cord (Fig. 1B), the major immunoreactive peak co-eluted with neuromedin K whereas in the caudate nucleus (Fig. 2A), substantia nigra (Fig. 2B) and hypothalamus (Fig. 3A) the major immunoreactive peak co-eluted with substance K. Furthermore, in the caudate nucleus (Fig. 2A) the amount of immunoreactive material
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EluhonTime (min)
Fig. 2. RP-HPLC chromatogram of (A) cat caudate nucleus and (B) cat substantia nigra. The column was eluted under the same conditions as described in Fig. 1. In addition to the synthetic standards, the oxidized forms of the peptides eluted at the following positions in this gradient system: substance K sulphoxide 5.5 rain; neuromedin K monosulphoxide 23.5 rain; neuromedin K disulphoxide 14.5 min. The major peak of KLI co-elutes with synthetic substance K in both the caudate nucleus and substantia nigra. A similar result was obtained in three separate experiments.
Fig. 3. RP-HPLC chromatogram of (A) cat hypothalamusand (B) cat terminal ileum. The column was eluted under the same conditions as described in Fig. 1. The synthetic standards and their sulphoxides eluted at the positions detailed in both Fig. 1 and Fig. 2. The major peak of KLI in the cat hypothalamus coelutes with substance K whereas in the cat ileum the major immunoreactive peak co-elutes with neuromedin K. A similar result was obtained in three separate experiments.
co-eluting with n e u r o m e d i n K was negligible. In p e r i p h e r a l tissues of the cat, K L I was d e t e c t e d in m o d e r a t e amounts in the pituitary but in only low concentrations in the dorsal r o o t ganglia. K L I was present t h r o u g h o u t the gastrointestinal tract with the highest concentration found in the d u o d e n u m (Table II). On R P - H P L C analysis of the cat ileum extract, the m a j o r i m m u n o r e a c t i v e p e a k co-eluted with synthetic n e u r o m e d i n K with only a negligible a m o u n t of K L I co-eluting with substance K (Fig. 3B). The distribution of K L I closely r e s e m b l e d that of substance P-like immunoreactivity (SPLI). However, although the ratio of SPLI to K L I varied in the tissues tested this was not found to be statistically significant (Tables I and II). The concentration of SPLI was at least 6-fold higher than that of K L I in the frontal cortex and c e r e b e l l u m , 4-fold higher in the dorsal root ganglion and b e t w e e n 1.5- and 3-fold higher in a
variety of o t h e r tissues including the hypothalamus, substantia nigra, brainstem, and t h r o u g h o u t the gas~ trointestinal tract. DISCUSSION In the cat brain, the regional distribution of KLI was similar to that found in the raft with the highest concentrations of K L I found in the substantia nigra followed by the h y p o t h a l a m u s and caudate nucleus. Similarly, only low amounts were d e t e c t e d in the cat frontal cortex and cerebellum. The concentrations of K L I in each area of the cat brain was if anything slightly lower than the corresponding values for the rat, although in the cat substantia nigra it was slightly higher. In p e r i p h e r a l cat tissues the concentrations of K L I were similar to those found in the raft. However, the distribution of K L I throughout the gastroin-
231 TABLE I
the concentrations of SPLI were usually between 2
Concentrations of kassinin-like (KL1) and substance P-like (SPL1) immunoreactivity in central tissues of the cat
and 3 times higher than those of KLI. In rat tissues, the concentration of SPLI was consistently twice that of KLI with only minor variation 7, Although tissue
Each value represents the mean _+ S.E.M. of between 4 and 9 observations. Region
KLI
SPL1
SPLI/KLI
(pmol/g wet weight)
Hypothalamus Thalamus Substantia nigra Caudate nucleus Olfactory bulb Frontal cortex Cerebellum Pons/Medulla Spinal cord
172 + 32 49 + 7 635 + 92 110 + 7 20 + 2 7 _+2 2_+0.3 52 _.9_5 65 -+ 9
331 _+ 19 135 _+ 16 1389 _+85 140 _+ 18 28 _+2 35 _+ 12 11_+2 130 + 25 126 _+28
concentrations of SPLI in the cat gastrointestinal tract were similar to previously published data 2, we have been unable to find a similar study on the detailed distribution of SPLI in the cat brain. Perhaps
2.2 _+0.4 2.8 _+0.1 1.8 _+0.2 1.5 _+0.3 1.5 _+0.2 6.3 _+2.5 6.4_+2.8 2.8 _+0.4 2.1 _+0.2
testinal tract was different from the rat with the cat d u o d e n u m and descending colon containing the highest concentrations of KLI. These tissues were found to have the lowest concentrations of KLI in the rat 7. The general distribution of KLI in the cat central and peripheral tissues, as in the cat, closely resembled the distribution of SLPI though a further a n o m a l y was in the ratio of SPLI to KLI. Thus, in certain areas of the cat brain the concentration of SPLI was at least 6
the most surprising differences between the cat and rat were found in the H P L C analysis of the various tissue extracts. In the rat we found that the kassininlike immunoreactive material co-eluting with substance K was consistently the major source of KLI in all tissues tested< In contrast, in cat tissue extracts, although the major source of KLI co-eluted with substance K in the caudate nucleus, substantia nigra and hypothalamus, the reverse situation was found in the spinal cord and ileum where the major peak of immunoreactivity co-eluted with n e u r o m e d i n K. Recently, the m R N A encoding the tachykinin precursor was isolated from bovine striatum 11. Surprisingly, although the sequences of substance P and substance K were present, that of n e u r o m e d i n K was missing. Nevertheless, the presence of these peptides at a genetic level raises the possibility that the differences
times that of KLI whilst in the gastrointestinal tract
observed between the relative a m o u n t s of substance K and n e u r o m e d i n K in the cat and rat spinal cord and ileum could well be due to gene expression.
TABLE II
Furthermore, it should also prove interesting to observe whether this inter-species variation in the ratio of substance K to n e u r o m e d i n K is found in their
Concentrations of kassinin-like (KL1) and substance P-like (SPL D immunoreactivity in peripheral tissues of the cat
Each value represents the mean _+ S.E.M. of between 3 and 5 observations; (n.d. = not determined). Region
KLI
SPL1
SPL1/KL1
Pituitary gland Superior cervical ganglion Dorsal root ganglion Adrenal gland Oesophagus Fundus Antrum Duodenum Jejunum Ileum Colon Pancreas
20.3 + 6.1
24.3 _+7.2
1.2 _+0.1
14.1 _+ 1.3 6.7 _+2.4 < 1 11,3 + 1.1 3.5 _+3.0 6.9 + 1.4 17.4 _+2.0 6.2 _+1.5 6.4 _+2.6 11.0_ + 1.2 1.5 _+0.2
n.d. 26.1 + 8.0 n.d. 31.0 + 4.7 7.6 _+0.9 16.3 _+0.9 40.1 _+8.8 11.8 _+0.4 9.9 _4=-3.2 32.1 _+8.2 n.d.
4.3 _+2.4 2.9 + 0.5 2.2 _+0.9 2.1 + 0.4 2.6 + 0.9 2.1 _+0.4 2.2 _+0.9 2.9+0.8
(pmol/g wet weight)
pharmacology with the possible existence of receptor subtypes. In conclusion, the present study shows that KLI is widely distributed throughout cat central and peripheral tissues where it closely resembles the distribution of SPLI. However, there exist important qualitative differences in the KLI found not only between certain areas of the cat but also between the cat and rat. ACKNOWLEDGEMENTS We thank Mrs. M. W y n n for typing the m a n u script. J.C.H. is a Medical Research Council Postdoctoral Fellow. This work was carried out during the tenure of a Postdoctoral Fellowship to J.E,M. from the Muscular Dystrophy Association.
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