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Modulation of endopeptidase activity by calcitonin gene related peptide: a mechanism affecting substance P action?
Biochimie 70 (1988) 65-68 ©Soci6t6 de Chimie biologique/Elsevier, Paris
65
Modulation of endopeptidase activity by calcitonin gene related peptide" a mechanism affecting substance P action? Fred NYBERG, Pierre LE GREVI~S and Lars TERENIUS
Department of Pharmacology, Box 591, 751 24 Uppsala, Sweden (Received 3-9-1987, accepted after revision 19-10-1987)
Summary -
Peptides with hormonal or neuronal activity are derived by enzymatic processing from pro-hormones, which by themselves are biologically inert. Processing and other enzymatic conversions may occur step-wise, leading to the formation of a cascade of biologically active (or inactive) peptides. The neurokin in substance P is known to be metabolically transformed both by amino- and endopeptidases. More N-terminal substance (1-7) has been found than C-terminal (2-11 to 5-11) fragments in various CNS areas. The substance P (1-7) fragment also shows biological activity e.g., providing analgesia, lowering blood pressure, inhibiting aggressive behavior and (in contrast to substance P) inhibiting grooming behavior. An endopeptidase generating substance P (1-7) and to a lesser extent, substance (1-8), has been isolated and characterized from human cerebrospinal fluid (CSF) and bovine spinal cord, as a metalloenzyme with essential SH-groups. Substance P co-exists with calcitonin gene related peptide (CGRP) in a large population of nonmyelinated primary afferent ('pain') fibers. Intrathecal injection of substance P causes behavioral and physiological responses which are potentiated and prolonged by CGRP. It was found that CGRP competes with substance P for the endopeptidase, it is suggested that the main action of CGRP in the spinal cord is to inhibit substance P degradation. caleitonin gene related peptide / endopeptisase / co-existence of peptides / substance P
Introduction
The tachykinin family of peptides comprises a large number of members characterized by structural homology of the C-terminals and certain common biological e'ffects, including contraction of non-vascular smooth muscle. Partly for historical reasons, the best known tachykinin is substance P. From a functional point of view, it is of particular interest that this peptide has been found in small diameter afferent ('pain') fibers [1]. Until recently, this tachykinin was the only one known to occur in mammalian tissue. Now, a whole family of tachykinins ofmammalian origin is Known. These pep-
tides have been isolated from nerve tissue collectively termed neurokinins. Despite their structural homology, the neurokinins act through several receptors [2]. It is likely that the structural determinants giving receptor selectivity are present within the N-terminal sequences, which are highly dissimilar (Table I shows structures of the most commonly studied neurokinins). These observations suggeg z that neurokinin N-terminal fragments lacking the common C-terminal sequence could have biological activity of their own. To investigate this possibility, several N-terminal fragments of substance P were tested for behavioral and other central nervous system (CNS) activities.
F. Nyberg et al.
66
Table I. Amino acid sequences of the mammalian tachykinins derived from pre-pro-tachykinin A and B genes [3]. Pre-pro-tachykinin A : Neurokinin A Substance P
His-Lys-Thr-Asp-Ser-Phe-VaI-Gly-Leu-Met-NH2 Arg-Pro-Lys-Pro-Gln-GIn-Phe-Phe-Gly-Leu-Met-NH2
Pre-pro-tachykinin B : Neurokinin B
Asp-Met-His-Asp-Phe-Phe-VaI-Gly-Leu-Met-NH2
Table il. Selected biological effects of substance P (SP), its N-terminal (1-7) fragment or its C-terminal (6-11) fragment. Behavioral test
Biological effect SP
SP (!-7)
SP (6-11)
Ref.
Hot plate
analgesia
analgesia
none
5
~llood-pressure (baro-receptor reflex)
lowered
lowered
none
6
Aggressive behavior (provocation)
inhibited
inhibited
increased
5
Grooming behavior
increased
inhibited
increased
5
The peptides were microinjected intracerebroventricularly into rats.
Substance P fragments and substance P fragmentation in nerve tissue
The (1-7) fragment was not only found to be active but also to possess activities which were partly from those of the parent compound or those of C-terminal fragments [4-6]. Certain representative activities are presented in Table II. These findings prompted investigation into the potential existence of substance P fragments in nerve tissue.
The metabolic degradation of substance P has been studied by several groups. In nerve tissue, degradation by aminopeptidases, endopeptidases or carboxypeptidases has been described. Since the tachykinins have an amidated and
Table !I!. Levels ofsubstance P (SP), SP (1-7) and SP carboxy-terminal fragments (SP-C) in areas ofthe rat brain and spinal cord (n = 6). SP
SP (1-7)
SP,C
Tissue (pmol/g wet weight) Brain hyoothalamus midbr;n medulla oblongata striatum
84 60 52 58
Spinal cord dorsal part ventral part
61 _+ 5.0 17 _+ 1.9
_+ 6.2 ___5.3 ___4.7 -+- 3.2
2.9 2.3 2.2 1.3
+ 0.34 ___0.42 __+0.13 ___0.17
5.8 ± 0.29 3. I _ 0.39
1.6 0.99 0.77 0.78
+__0.18"* + 0.11" ___0.06*** _4_0.04
0.98 ----.0.07*** 0.78 ___0.16"**
From I111. *p
Modulation of substance P endopeptidase therefore metabolically protected C-terminus, most interest has been focused on aminopeptidases [7, 8] and endopeptidases [9, 10]. An indirect approach to the metabolic fragmentation routes is to measure the actual levels of substance P metabolites in nerve tissue. By radioimmunoassay, substance P (1-7) as well as C-terminal (2-11 to 5-11) fragments could be determined separately from the parent compound. Quite unexpectedly, it was found [11] that substance P (!-7) levels were always higher than C-terminal fragments in the CNS. This was particularly evident and significant in the spinal cord, the medulla oblongata and the hypothalamus (Table III). The regional differences in relative substance P (1-7) content suggest that the importance of this transformation differs in various areas of the CNS, and indirectly points to a functional importance of this metabolic pathway. Since substance P (1-7) is present in h u m a n cerebrospinal fluid (CSF) [12], it was investigated and could also be confirmed that endopeptidase activity capable of generating the fragm e n t was present in the CSF [13]. The enzyme generated 1.3 to 1.4 times higher molar a m o u n t s of (1-7) than (1-8) fi'agment and was sensitive to EDTA and dithiothreitol, indicating that it is a metalloenzyme with essential SI-/-groups. It was not affected by inhibitors of enkephalinase or angiotensin converting enzyme. The molecular weight is around 43000 [!3]. Structure-activity comparisons showed the enzyme to have less activity against substance P (3-11), even less activity against substance P (5-11) and no activity against neurokinins A and B (Table IV), substance P (1-8), Leu-enkephalin and fl-casomorphin. An enzyme with similar properties can be isolated from bovine spinal cord (work in progress). Preliminary studies indicate that the enzyme is bound to neuronal membranes. The enzyme has properties similar to the enzyme described by Lee et al. [10], although they report cleavage also at the 6-7 bend. This, however, may be
67
due to an impurity, since during progressive purification, the formation of SP (1-6) is reduced relative to SP (1-7) and SP (1-8).
Table IV. Conversion of substance P and related tachykinins by endopeptidase from human CSF. Peptide
% Conversiona
Substance P
90
Substance P (5-11)
10
Neurokinin A
<5
Neurokinin B
<5
a Incubations were carried out at 37°C and pH 7.8 with 5 nmol of peptide and 5 ~g of enzyme. HPLC analysis was used to monitor enzymatic conversion.
Interactions between the metabolism of substance P and that of caicitonin gene related peptide (CGRP) The calcitonin gene undergoes tissue specific splicing [14]. In nerve tissue, the pro-hormone contains a polypeptide called calcitonin gene related peptide (CGRP). This peptide occurs in a large proportion of non-myelinated afferent C-fibers, partly in the same nerves as substance P° The extension of this system is at least as large as that of substance P [15, 16], The coexistence of substance P and CGRP suggests that they also shown functional interactions. By intrathecal injection into rats, substance P is known to produce a caudally directed 'scratching and biting' behavior, suggesting an irritant effect or even pain [1], commensurate with its putative role as a transmitter in 'pain" fibers. The effect of substance P is dose-dependent; CGRP however, does not produce a comparable response. If substance P and CGRP were injected together, to mimick what may be occuring naturally, CGRP was found to markedly prolong the effects of substance P and
Table V. Sequence alignment of substance P and hCGRP in relation to principal sites of cleavage by endopeptidase. Substance P
Arg-Pro-Lys- Pro-G In-Gin - Phe- Phe-G ly-Leu-Met-N H2
hCGRP (ll-21)
His-Arg-Leu-Ala-Gly-Leu-Leu-Ser-Arg-Ser-Giy
Arrows denote bonds cleaved.
68
F. Nyberg et al.
also to potentiate the magnitude of the response [15]. An analogous observation was made using a physiological response, i.e., recordings from flexor a-motoneurons in the spinal cord. Here, intratiaecal CGRP injection produced some effect at very high does and it very markedly prolonged and potentiated the effects of substance P. Sural nerve stimulation caused responses indistinguishable from those of substance P; CGRP was able to prolong and potentiate these responses to sural nerve stimulation as well [17]. One possible mechanism for the potentiation of the substance P-induced effect is an interaction with a common metabolic degradation pathway. Since the substance P (1-7) fragments does not have typical substance P activity in the spinal cord, the degradation of the parent compound to the fragment is an inactivation pathway. When tested for interaction, it was indeed found that CGRP competitively inhibits substance P degradation [18]. More recent studies have shown that the enzyme cleaves human (h) CGRP at the (17-18) bond with an affinity equal to that for substance P (unpublished observations). There is no sequence homology of these two peptides in general or in sequence around the cleavage sites (Table V). However, conformational analysis suggests similarities in tertiary structure. The substance P (4-8) fragment adopts an ahelical structure in water, stabilized by an interaction between the C-terminal carboxa_m__ide with the primary amide from both glutamines [19]. Molecular modelling of the CGRP molecule shows that the (8-28) sequence can be considered an amphiphilic a-helix [20]. As in substance P, hydrophilic domains consist mainly of neutral or basic amino acids. These structural considerations may also explain why substance P (5-11) and other C-terminal fragments are less effectively degraded by the enzyme than the parent compound (cf. Table IV). Substance P, but not its N-terminal fragments, is known to have transmitter-like characteristics in primary afferent fibers. Electric stimulation produces a massive release of substance P, an effect blocked by morphine [21]. Also, CGRP is released by somatic stimulation [14] and it is likely that it is co-released with substance P. Since CGRP alone appears to be physiologically and behaviorally much less active than substance P, its strong interaction with the latter, may be its main function. Also
questioning the direct action of CGRP in nerve tissue [22] is the equivocal existence of CGRP binding sites ('receptors') in the dorsal spinal cord. In conclusion, two structurally unrelated peptides, substance P and CGRP, which may also co-exist in the same neurons, are both degraded by the same endopeptidase. It is suggested that this is a basis for functional interactions observed between these peptides.
Acknowledgments This work was supported by the Swedish Medical Research C o u n c i l and M a g n u s Bergvalls Stiftelse.
References 1. Pernow B. (1983) Phannacol. Rev. 35, 85-141 2. Lee C.-M., Campbell N.J., Williams B.J. & Iversen L.L. (1986) Eur. J. PharmacoL 130, 209-217 3. Kotani H., Hoshimaru M., Nawa H. & Nakanishi S. (1986) Proc. Natl. Acad. Sci. USA 83, 7074-7078 4. Stewart J.M., Hall M.E., Harkins J., Frederickson R.C.A., Terenius L., H6kfelt T. & Krivoy W.A. (1982) Peptides 3, 851-857 5. Hall M.E. & Stewart J.M. (1983) Peptides 4, 763-768 6. Hall M.E., Miley F.B. & Stewart J.M. (1987) L(/'eSci. 40, 1909-1914 7. Orlowski M., Wilk E., Pearce S. & Wilk S. (1979) J. Neurochem. 33, 461-469 8. Biumberg S., Teichberg V.I., Charli J.L., Hersh L.B. & McKelvy J.F. (1980) Brain Res. 192, 477-486 9. Benuck M. & Marks N. (1975) Biochem. Biophys. Res. Commun. 65, 153-159 10. Lee C.-M., Sandberg B.E.B., Hanley M.R. & Iversen L.L. (1981) Eur. J. Biochem. 114, 315-327 i 1. Sakurada T., Le Grev6s P., Stewart J.M. & Terenius L. (1985) J. Ncr~rochem. 44, 718-722 12. Rim6n R., Le Grev6s P., Nyberg F., Heikkilii L., Salmela L, & Terenius L. (1984) Biol. Psychiatry 19, 509516 13. Nyberg F., Le Grev6s P., Sundqvist C. & Terenius L. (1984) Biochem. B~ophys. Res. Commun. 125, 244-250 14. Rosenfeld M.G., Mermod J.-J., Amara S.G., Swanson L.W., Sawchenko P.E., Rivier J., Vale W.W. & Evans R.M. (1983) Nature 304, 129-135 15. Wiesenfeid-Hallin Z., H6kfelt T., Lundberg J.M., Forssmann W.G., Reinecke M., Tschopp F.A. & Fischer J.A. (1984) Neurosci. Lett. 52, 199-204 16. Tschopp F.A., Henke H., Petermann J.B., Tobler P.H., Janzer R., H6kfelt T., Lundberg J.M. & Fischer J.A. (1985) Proc. Natl. Acad. Sci USA 82, 248-252 17. WoolfC. & Wiesenfeld-Hallin Z. (1986) Neurosci. Lett. 66, 226-230 18. Le Grev6s P., Nyberg F., Terenius L. & H6kfelt T. (1985) Eur. J. Pharmacol. 115, 309-311 19. Chassaing G., Convert O. & Lavielle S. (1986) Eur. J. Biochem. 154, 77-85 20. Taylor J.W. & Kaiser E.T. (1986) Pharmacol. Rev. 38, 291-319 21. Yaksh T.L., Jessell T.M., Gamse R., Mudge A.W. & Leeman S.E. (1980) Nature 286, 155-156 22. Hfkfelt T. & Terenius L. (1987) Trends Neuros¢'i. 10, 22-23
65
Modulation of endopeptidase activity by calcitonin gene related peptide" a mechanism affecting substance P action? Fred NYBERG, Pierre LE GREVI~S and Lars TERENIUS
Department of Pharmacology, Box 591, 751 24 Uppsala, Sweden (Received 3-9-1987, accepted after revision 19-10-1987)
Summary -
Peptides with hormonal or neuronal activity are derived by enzymatic processing from pro-hormones, which by themselves are biologically inert. Processing and other enzymatic conversions may occur step-wise, leading to the formation of a cascade of biologically active (or inactive) peptides. The neurokin in substance P is known to be metabolically transformed both by amino- and endopeptidases. More N-terminal substance (1-7) has been found than C-terminal (2-11 to 5-11) fragments in various CNS areas. The substance P (1-7) fragment also shows biological activity e.g., providing analgesia, lowering blood pressure, inhibiting aggressive behavior and (in contrast to substance P) inhibiting grooming behavior. An endopeptidase generating substance P (1-7) and to a lesser extent, substance (1-8), has been isolated and characterized from human cerebrospinal fluid (CSF) and bovine spinal cord, as a metalloenzyme with essential SH-groups. Substance P co-exists with calcitonin gene related peptide (CGRP) in a large population of nonmyelinated primary afferent ('pain') fibers. Intrathecal injection of substance P causes behavioral and physiological responses which are potentiated and prolonged by CGRP. It was found that CGRP competes with substance P for the endopeptidase, it is suggested that the main action of CGRP in the spinal cord is to inhibit substance P degradation. caleitonin gene related peptide / endopeptisase / co-existence of peptides / substance P
Introduction
The tachykinin family of peptides comprises a large number of members characterized by structural homology of the C-terminals and certain common biological e'ffects, including contraction of non-vascular smooth muscle. Partly for historical reasons, the best known tachykinin is substance P. From a functional point of view, it is of particular interest that this peptide has been found in small diameter afferent ('pain') fibers [1]. Until recently, this tachykinin was the only one known to occur in mammalian tissue. Now, a whole family of tachykinins ofmammalian origin is Known. These pep-
tides have been isolated from nerve tissue collectively termed neurokinins. Despite their structural homology, the neurokinins act through several receptors [2]. It is likely that the structural determinants giving receptor selectivity are present within the N-terminal sequences, which are highly dissimilar (Table I shows structures of the most commonly studied neurokinins). These observations suggeg z that neurokinin N-terminal fragments lacking the common C-terminal sequence could have biological activity of their own. To investigate this possibility, several N-terminal fragments of substance P were tested for behavioral and other central nervous system (CNS) activities.
F. Nyberg et al.
66
Table I. Amino acid sequences of the mammalian tachykinins derived from pre-pro-tachykinin A and B genes [3]. Pre-pro-tachykinin A : Neurokinin A Substance P
His-Lys-Thr-Asp-Ser-Phe-VaI-Gly-Leu-Met-NH2 Arg-Pro-Lys-Pro-Gln-GIn-Phe-Phe-Gly-Leu-Met-NH2
Pre-pro-tachykinin B : Neurokinin B
Asp-Met-His-Asp-Phe-Phe-VaI-Gly-Leu-Met-NH2
Table il. Selected biological effects of substance P (SP), its N-terminal (1-7) fragment or its C-terminal (6-11) fragment. Behavioral test
Biological effect SP
SP (!-7)
SP (6-11)
Ref.
Hot plate
analgesia
analgesia
none
5
~llood-pressure (baro-receptor reflex)
lowered
lowered
none
6
Aggressive behavior (provocation)
inhibited
inhibited
increased
5
Grooming behavior
increased
inhibited
increased
5
The peptides were microinjected intracerebroventricularly into rats.
Substance P fragments and substance P fragmentation in nerve tissue
The (1-7) fragment was not only found to be active but also to possess activities which were partly from those of the parent compound or those of C-terminal fragments [4-6]. Certain representative activities are presented in Table II. These findings prompted investigation into the potential existence of substance P fragments in nerve tissue.
The metabolic degradation of substance P has been studied by several groups. In nerve tissue, degradation by aminopeptidases, endopeptidases or carboxypeptidases has been described. Since the tachykinins have an amidated and
Table !I!. Levels ofsubstance P (SP), SP (1-7) and SP carboxy-terminal fragments (SP-C) in areas ofthe rat brain and spinal cord (n = 6). SP
SP (1-7)
SP,C
Tissue (pmol/g wet weight) Brain hyoothalamus midbr;n medulla oblongata striatum
84 60 52 58
Spinal cord dorsal part ventral part
61 _+ 5.0 17 _+ 1.9
_+ 6.2 ___5.3 ___4.7 -+- 3.2
2.9 2.3 2.2 1.3
+ 0.34 ___0.42 __+0.13 ___0.17
5.8 ± 0.29 3. I _ 0.39
1.6 0.99 0.77 0.78
+__0.18"* + 0.11" ___0.06*** _4_0.04
0.98 ----.0.07*** 0.78 ___0.16"**
From I111. *p
Modulation of substance P endopeptidase therefore metabolically protected C-terminus, most interest has been focused on aminopeptidases [7, 8] and endopeptidases [9, 10]. An indirect approach to the metabolic fragmentation routes is to measure the actual levels of substance P metabolites in nerve tissue. By radioimmunoassay, substance P (1-7) as well as C-terminal (2-11 to 5-11) fragments could be determined separately from the parent compound. Quite unexpectedly, it was found [11] that substance P (!-7) levels were always higher than C-terminal fragments in the CNS. This was particularly evident and significant in the spinal cord, the medulla oblongata and the hypothalamus (Table III). The regional differences in relative substance P (1-7) content suggest that the importance of this transformation differs in various areas of the CNS, and indirectly points to a functional importance of this metabolic pathway. Since substance P (1-7) is present in h u m a n cerebrospinal fluid (CSF) [12], it was investigated and could also be confirmed that endopeptidase activity capable of generating the fragm e n t was present in the CSF [13]. The enzyme generated 1.3 to 1.4 times higher molar a m o u n t s of (1-7) than (1-8) fi'agment and was sensitive to EDTA and dithiothreitol, indicating that it is a metalloenzyme with essential SI-/-groups. It was not affected by inhibitors of enkephalinase or angiotensin converting enzyme. The molecular weight is around 43000 [!3]. Structure-activity comparisons showed the enzyme to have less activity against substance P (3-11), even less activity against substance P (5-11) and no activity against neurokinins A and B (Table IV), substance P (1-8), Leu-enkephalin and fl-casomorphin. An enzyme with similar properties can be isolated from bovine spinal cord (work in progress). Preliminary studies indicate that the enzyme is bound to neuronal membranes. The enzyme has properties similar to the enzyme described by Lee et al. [10], although they report cleavage also at the 6-7 bend. This, however, may be
67
due to an impurity, since during progressive purification, the formation of SP (1-6) is reduced relative to SP (1-7) and SP (1-8).
Table IV. Conversion of substance P and related tachykinins by endopeptidase from human CSF. Peptide
% Conversiona
Substance P
90
Substance P (5-11)
10
Neurokinin A
<5
Neurokinin B
<5
a Incubations were carried out at 37°C and pH 7.8 with 5 nmol of peptide and 5 ~g of enzyme. HPLC analysis was used to monitor enzymatic conversion.
Interactions between the metabolism of substance P and that of caicitonin gene related peptide (CGRP) The calcitonin gene undergoes tissue specific splicing [14]. In nerve tissue, the pro-hormone contains a polypeptide called calcitonin gene related peptide (CGRP). This peptide occurs in a large proportion of non-myelinated afferent C-fibers, partly in the same nerves as substance P° The extension of this system is at least as large as that of substance P [15, 16], The coexistence of substance P and CGRP suggests that they also shown functional interactions. By intrathecal injection into rats, substance P is known to produce a caudally directed 'scratching and biting' behavior, suggesting an irritant effect or even pain [1], commensurate with its putative role as a transmitter in 'pain" fibers. The effect of substance P is dose-dependent; CGRP however, does not produce a comparable response. If substance P and CGRP were injected together, to mimick what may be occuring naturally, CGRP was found to markedly prolong the effects of substance P and
Table V. Sequence alignment of substance P and hCGRP in relation to principal sites of cleavage by endopeptidase. Substance P
Arg-Pro-Lys- Pro-G In-Gin - Phe- Phe-G ly-Leu-Met-N H2
hCGRP (ll-21)
His-Arg-Leu-Ala-Gly-Leu-Leu-Ser-Arg-Ser-Giy
Arrows denote bonds cleaved.
68
F. Nyberg et al.
also to potentiate the magnitude of the response [15]. An analogous observation was made using a physiological response, i.e., recordings from flexor a-motoneurons in the spinal cord. Here, intratiaecal CGRP injection produced some effect at very high does and it very markedly prolonged and potentiated the effects of substance P. Sural nerve stimulation caused responses indistinguishable from those of substance P; CGRP was able to prolong and potentiate these responses to sural nerve stimulation as well [17]. One possible mechanism for the potentiation of the substance P-induced effect is an interaction with a common metabolic degradation pathway. Since the substance P (1-7) fragments does not have typical substance P activity in the spinal cord, the degradation of the parent compound to the fragment is an inactivation pathway. When tested for interaction, it was indeed found that CGRP competitively inhibits substance P degradation [18]. More recent studies have shown that the enzyme cleaves human (h) CGRP at the (17-18) bond with an affinity equal to that for substance P (unpublished observations). There is no sequence homology of these two peptides in general or in sequence around the cleavage sites (Table V). However, conformational analysis suggests similarities in tertiary structure. The substance P (4-8) fragment adopts an ahelical structure in water, stabilized by an interaction between the C-terminal carboxa_m__ide with the primary amide from both glutamines [19]. Molecular modelling of the CGRP molecule shows that the (8-28) sequence can be considered an amphiphilic a-helix [20]. As in substance P, hydrophilic domains consist mainly of neutral or basic amino acids. These structural considerations may also explain why substance P (5-11) and other C-terminal fragments are less effectively degraded by the enzyme than the parent compound (cf. Table IV). Substance P, but not its N-terminal fragments, is known to have transmitter-like characteristics in primary afferent fibers. Electric stimulation produces a massive release of substance P, an effect blocked by morphine [21]. Also, CGRP is released by somatic stimulation [14] and it is likely that it is co-released with substance P. Since CGRP alone appears to be physiologically and behaviorally much less active than substance P, its strong interaction with the latter, may be its main function. Also
questioning the direct action of CGRP in nerve tissue [22] is the equivocal existence of CGRP binding sites ('receptors') in the dorsal spinal cord. In conclusion, two structurally unrelated peptides, substance P and CGRP, which may also co-exist in the same neurons, are both degraded by the same endopeptidase. It is suggested that this is a basis for functional interactions observed between these peptides.
Acknowledgments This work was supported by the Swedish Medical Research C o u n c i l and M a g n u s Bergvalls Stiftelse.
References 1. Pernow B. (1983) Phannacol. Rev. 35, 85-141 2. Lee C.-M., Campbell N.J., Williams B.J. & Iversen L.L. (1986) Eur. J. PharmacoL 130, 209-217 3. Kotani H., Hoshimaru M., Nawa H. & Nakanishi S. (1986) Proc. Natl. Acad. Sci. USA 83, 7074-7078 4. Stewart J.M., Hall M.E., Harkins J., Frederickson R.C.A., Terenius L., H6kfelt T. & Krivoy W.A. (1982) Peptides 3, 851-857 5. Hall M.E. & Stewart J.M. (1983) Peptides 4, 763-768 6. Hall M.E., Miley F.B. & Stewart J.M. (1987) L(/'eSci. 40, 1909-1914 7. Orlowski M., Wilk E., Pearce S. & Wilk S. (1979) J. Neurochem. 33, 461-469 8. Biumberg S., Teichberg V.I., Charli J.L., Hersh L.B. & McKelvy J.F. (1980) Brain Res. 192, 477-486 9. Benuck M. & Marks N. (1975) Biochem. Biophys. Res. Commun. 65, 153-159 10. Lee C.-M., Sandberg B.E.B., Hanley M.R. & Iversen L.L. (1981) Eur. J. Biochem. 114, 315-327 i 1. Sakurada T., Le Grev6s P., Stewart J.M. & Terenius L. (1985) J. Ncr~rochem. 44, 718-722 12. Rim6n R., Le Grev6s P., Nyberg F., Heikkilii L., Salmela L, & Terenius L. (1984) Biol. Psychiatry 19, 509516 13. Nyberg F., Le Grev6s P., Sundqvist C. & Terenius L. (1984) Biochem. B~ophys. Res. Commun. 125, 244-250 14. Rosenfeld M.G., Mermod J.-J., Amara S.G., Swanson L.W., Sawchenko P.E., Rivier J., Vale W.W. & Evans R.M. (1983) Nature 304, 129-135 15. Wiesenfeid-Hallin Z., H6kfelt T., Lundberg J.M., Forssmann W.G., Reinecke M., Tschopp F.A. & Fischer J.A. (1984) Neurosci. Lett. 52, 199-204 16. Tschopp F.A., Henke H., Petermann J.B., Tobler P.H., Janzer R., H6kfelt T., Lundberg J.M. & Fischer J.A. (1985) Proc. Natl. Acad. Sci USA 82, 248-252 17. WoolfC. & Wiesenfeld-Hallin Z. (1986) Neurosci. Lett. 66, 226-230 18. Le Grev6s P., Nyberg F., Terenius L. & H6kfelt T. (1985) Eur. J. Pharmacol. 115, 309-311 19. Chassaing G., Convert O. & Lavielle S. (1986) Eur. J. Biochem. 154, 77-85 20. Taylor J.W. & Kaiser E.T. (1986) Pharmacol. Rev. 38, 291-319 21. Yaksh T.L., Jessell T.M., Gamse R., Mudge A.W. & Leeman S.E. (1980) Nature 286, 155-156 22. Hfkfelt T. & Terenius L. (1987) Trends Neuros¢'i. 10, 22-23