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The effect of galanin, CGRP and ANP on spontaneous smooth muscle activity of rat uterus
Peptides, Vol. 9, pp. 497-500. ©Pergamon Press plc, 1988. Printed in the U.S.A.
01%-9781/88 $3.00 + .00
The Effect of Galanin, CGRP and ANP on Spontaneous Smooth Muscle Activity of Rat Uterus TOKE BEK,* BENT OTTESENt
AND JAN FAHRENKRUG*
Department of Clinical Chemistry, Bispebjerg Hospital, DK-2400 Copenhagen, NV, Denmark and tDepartment o f Gynaecology and Obstetrics, Hvidovre Hospital DK-2650 Hvidovre, Denmark R e c e i v e d 31 A u g u s t 1987 BEK, T., B. OTTESEN AND J. FAHRENKRUG. The effect of galanin, CGRPand ANP on spontaneous smooth muscle activity of rat uterus. PEPTIDES 9(3)497-500, 1988.--In the present study the effect of the newly isolated peptides galanin, calcitonin gene-related peptide (CGRP) and atrial natriuretic peptide (ANP) was examined on spontaneous uterine smooth muscle activity on the rat in vitro. Galanin showed a slight and insignificant stimulatory effect on the amplitude, while both CGRP and ANP were found to be potent inhibitors of the uterine smooth muscle contractions. In connection with the recent demonstration of galanin and CGRP nerves in the genital tract, these pharmacological findings suggest that the peptides may participate in the control of nervous control of uterine muscular activity, although the exact physiological roles remain to be clarified. Neuropeptides Uterine motility Atrial natriuretic peptide (ANP)
Galanin
Calcitonin gene-related peptide (CGRP)
METHOD
A N increasing number of peptides, originally isolated from extragenital sources, have by radioimmunoassay and immunocytochemistry been demonstrated in the genitourinary tract [9]. Recently two newly isolated peptides, galanin and CGRP, have been demonstrated in the female genital tract [2,4]. Galanin is a 29 amino acid peptide, originally isolated from porcine small intestine [12]. In the urogenital system of man and rat galanin immunoreactive nerve fibres are found within smooth muscle and in close relationship to blood vessels [2]. Calcitonin gene-related peptide (CGRP) consists of 37 amino acid residues [11]. This peptide, which has been identified in central and peripheral neural tissues, is a product of m R N A formed by alternative tissue-specific RNA splicing after transcription of the calcitonin gene [1 ]. In the rat genitourinary tract CGRP nerve fibres are associated mainly with blood vessels, non-vascular smooth muscle and epithelial cells [4]. In the uterus CGRP immunoreactive fibres are mainly distributed in the myometrium [4]. Atrial natriuretic peptide (ANP), a 28 amino acid peptide originally isolated from atrial muscle of the heart, has been shown to influence vascular and non-vascular smooth muscle activity [3]. In order to investigate a possible physiological role of the three peptides, galanin, CGRP and A N P in the female genital tract, we examined their effect on the activity of smooth muscle strips from rat uterus.
Animals Female non-pregnant Wistar rats weighing approximately 200 g were employed. Three days before sacrifice the animals received daily injections of oestradiolbenzoate, Ovex ®IM (Loeven, Denmark) (1.25 mg/kg) in order to obtain a spontaneous and rhythmic activity of the uterine musculature.
Tissue Strips On the day of experiment the animals were anaesthetised by intraperitoneal administration of pentobarbitone (1 g/kg). The uterus was immediately removed to ice-cold modified Krebs buffer with the following composition: NaC1 118 mmol/l, KCI 4.4 mmol/1, MgSO4 0.12 mmol/1, KH2PO3 1.2 mmol/l, KC1 2.51 mmol/1, NaHCO3 25 mmol/1 and glucose 11 mmol/1. Tissue strips measuring 1 × 1 × 10 mm were excised and mounted vertically between a horizontal bolt and an isometric force-displacement transducer (Grass FT03C, Grass Instruments, USA) in a 5 ml organ bath containing Krebs buffer thermostatically kept at 37.0°C. The buffer was continuously aerated with a mixture of 95% Oz and 5% CO2. The strips were subjected to tension of approximately 1 g and allowed to equilibrate until spontaneous contractions developed. The contractions were monitored on a linearecorder (Servogor RE 511, Goerz, Austria).
497
498
BEK, O T T E S E N AND F A H R E N K R U G
Galanin A
~
B
5 min.
120-
m
813
~
t
t~ tO
10-10 M
N
1'1
;
10
;
7
t,,-
8
- log conc. M
FIG. 1. (A) Shows the effect of increasing concentrations of galanin on myometrial strips from non-pregnant rat uterus in vitro. Amplitude (dashed line); frequency (solid line). Values (mean+SEM) are percentage change of the spontaneous contractions (n=6). (B) Shows a representative trace. (The short-lived aberrant contraction immediately after application of peptide is an artefact.) ANP
A
B
100I
._Z, .>
5 min.
80o (-
t't .¢- l
60"
E ._c 40-
~' Tt
t 10..8M
O} c'-
.g
20"
3 0
-log cone. M FIG. 2. (A) Shows the effect of increasing concentrations of ANP on myometrial strips from nonpregnant rat uterus in vitro. Amplitude (dashed line); frequency (solid line). Values (mean-+SEM) are percentage change of the spontaneous contractions (n =4). The amplitude was significantly reduced at l0 -9 tool/1 and above. (B) Shows a representative trace.
Experimental Procedure When a regular rhythm of contractions had developed, the dose-response relationship of each peptide was examined by adding ten-fold increasing concentrations to the organ bath. Application of peptide to the strips in a non-relaxed phase induced an initial short-lived and aberrant contraction. The next concentration of peptide was added when activity had returned to basal and at least identical contractions had appeared. The effect of porcine galanin was investigated in a concentration range from 10-11 to 10-7 mol/l, rat CGRP from 10-9 to 10-6 mol/l and rat ANP from 10-11 to 10-~ mol/1. Full dose-response curves for each peptide were established in strips from 4--6 animals. Peptides were synthetic, purchased from Peninsula Laboratories (St. Helens, United Kingdom).
Each tissue strip was its own control. Thus the effect on frequency and amplitude was compared to the frequency and amplitude immediately before peptide application. The effect was expressed as a percent change of the preceding control activity. Results are expressed as means-+-SEM. Statistical analysis was performed using an ANOVA test and in case of significant effect followed by a Newman-Keuls' test. Differences resulting in p-values less than 0.05 were considered significant. RESULTS
Galanin showed a slight and insignificant stimulatory el-
G A L A N I N , CGRP, ANP AND RAT U T E R U S
499
C(3RP
TABLE 1 DOSE-DEPENDENT INCREASEIN THE DURATIONOF INHIBITION OF SPONTANEOUSUTERINEMUSCLE ACTIVITYAFTER APPLICATIONOF ANP AND CGRP (MEAN -+ SEM)
5 min. t
I
Duration of Action (min) Concentration (mol/1) 10-11 10-1° 10-9 10-s 3×10 -8 10-7 3×10 7 10-6
ANP (n=4) 0 2.6 3.2 4.2
-+ 0 -+ 0.9 - 1.2 ± 1.7
4.9 ± 0.2 7.4 ± 1.1
CGRP (n=6)
0 --- 0 1.5 ± 1.0 7.0 ± 1.2 8.5 ± 3.7 16.6 --- 4.9 Total Inhibition
'I 3 x 10-8 M
FIG. 3. Representative trace showing CGRP-induced inhibition of spontaneous activity of myometrial strips from non-pregnant rat uterus. fect on the amplitude of uterine smooth muscle activity, while the frequency was unaffected (Fig. 1). ANP displayed a pronounced inhibitory action on the amplitude of spontaneous uterine contractions. The frequency, however, did not change significantly (Fig. 2). The duration of ANP action until total recovery increased moderately in a dosedependent manner (Table 1). Concentrations of CGRP 10-s mold completely inhibited spontaneous uterine contractions, while concentrations below this value had virtually no effect (Fig. 3). A prominent feature of this peptide was the extensive dose-related increase in duration of inhibition of spontaneous activity (Table 1). DISCUSSION
The present study provides evidence that CGRP, ANP and to a slight degree galanin influence spontaneous smooth muscle activity of the rat uterus. The purpose of pretreating the rats with oestradiolbenzoate was to ensure an estrogen dominated myometrium in all animals and thereby obtaining spontaneous and rhythmic activity of the smooth muscle specimens. Furthermore, this treatment produces identical estrogen levels in all rats, which is desirable since it has been shown that sex steroids are able to influence the susceptibility of smooth musculature to the effect of peptides for example vasoactive intestinal peptide [8]. The modest stimulatory effect of galanin on the amplitude contrasts to the pronounced ability of the peptide to induce contractions of smooth muscle preparations from the gastrointestinal tract and urinary bladder of the rat [12]. While the action and the presence of galanin im-
munoreactivity in the intestinal [5,10] and the urinary tracts [2] suggest that galanin participates in the autonomic nervous control of smooth muscle activity, the role of the peptide in uterine contractility seems questionable. The effect of CGRP was all or none in nature with regard to amplitude of spontaneous muscle activity in the rat uterus, whereas the duration of the inhibitory action increased dose-dependently. The nature of the response to CGRP is not really indicative for a physiological role as efferent neurotransmitter. It has, however, been suggested that CGRP in the genitourinary tract is present in non-myelinated sensory nerve fibres based on the fact that capsaicin pretreatment resulted in a substantial depletion of the CGRP content [4]. ANP was, like CGRP, found to be an inhibitor of uterine smooth muscle activity, and the inhibitory effect of ANP on amplitude was dose-related. At present, ANP has not yet been demonstrated in peripheral neurons and a role for ANP as a local neurotransmitter remains to be clarified. The concentrations of ANP needed to influence uterine smooth muscle activity are above the reported circulating plasma levels. Future studies will probably elucidate the possible role of ANP in the physiological control of the uterine activity. The regulation of uterine smooth muscle activity is complex. Besides the role of the classical adrenergic and cholinergic nerves, VIP and substance P have previously been shown to be inhibitory and stimulatory transmitters, respectively [6,7]. The present findings contribute to make the picture of peptidergic regulation of uterine smooth muscle activity even more complex.
REFERENCES
1. Amara, S. G., V. Jonas, M. G. Rosenfeld, E. S. Ong and R. M. Evans. Alternative RNA processing in calcitonin gene expression generates mRNA encoding different polypeptide products. Nature 298: 240-244, 1982. 2. Bauer, F. E., N. D. Christofides, G. W. Hacker, M. A. Blank, J. M. Polak and S. R. Bloom. Distribution of galanin immunoreactivity in the genitourinary tract of man and rat. Peptides 7: 5-10, 1986. 3. Cantin, M. and J. Genest. The heart and the atrial natriuretic factor. Endocr Rev 6: 107-127, 1986.
4. Ghatei, M. A., P. K. Mulderry, M. A. Blank, J. M. Allen, J. F. B. Morrison, J. M. Polak and S. R. Bloom. Calcitonin generelated peptide in the female rat urogenital tract. Peptides 6: 809-815, 1985. 5. Melander, T., T. Hfkfelt, A. ROkaeus, J. Fahrenkrug, K. Tatemoto and V. Mutt. Distribution of galanin-like immunoreactivity in the gastrointestinal tract of several mammalian species. Cell Tissue Res 239: 253-270, 1980. 6. Ottesen, B. Vasoactive intestinal polypeptide as neurotransmitter in the female genital tract. Am J Obstet Gynecol 147: 208224, 1983.
500 7. Ottesen, B., B. R. Gram and J. Fahrenkrug. Neuropeptides in the female genital tract: Effect on vascular and non-vascular smooth muscle. Peptides 4: 387-392, 1983. 8. Ottesen, B., J.-J. Larsen, P. Staun-Olsen, S. Gammeltoft and J. Fahrenkrug. Influence of pregnancy and sex steroids on concentration, motor effect and receptor binding and VIP in the rabbit female genital tract. Regul Pept 11: 83-92, 1985. 9. Ottesen, B., G. Wagner and J. Fahrenkrug. Peptidergic innervation of sexual organs. In: Handbook of Sexology. Vol 6, edited by J. M. A. Sitsen. Amsterdam: Elsevier, 1988, in press.
BEK, OTTESEN AND FAHRENKRUG 10. R6kaeus, A., T. Melander, T. H6kfelt, J. M. Lundberg, K. Tatemoto, M. Carlquist and V. Mutt. A galanin-like peptide in the central nervous system and intestine of the rat. Neurosci Lett 47: 161-166, 1984. 11. Rosenfeld, M. G., J.-J. Mermord, S. G. Amara, L. W. Swanson, P. E. Sawchenko, J. Rivier, W. W. Vale and R. M. Evans. Production of a novel neuropeptide encoded by the calcitonin gene via tissue specific RNA processing. Nature 304: 12%135, 1983.
12. Tatemoto, K., A. R6kaeus, H. J6rnvall, T. J. McDonald and V. Mutt. Galanin--A novel biologically active peptide from porcine intestine. FEBS Lett 164: 124-128, 1983.
01%-9781/88 $3.00 + .00
The Effect of Galanin, CGRP and ANP on Spontaneous Smooth Muscle Activity of Rat Uterus TOKE BEK,* BENT OTTESENt
AND JAN FAHRENKRUG*
Department of Clinical Chemistry, Bispebjerg Hospital, DK-2400 Copenhagen, NV, Denmark and tDepartment o f Gynaecology and Obstetrics, Hvidovre Hospital DK-2650 Hvidovre, Denmark R e c e i v e d 31 A u g u s t 1987 BEK, T., B. OTTESEN AND J. FAHRENKRUG. The effect of galanin, CGRPand ANP on spontaneous smooth muscle activity of rat uterus. PEPTIDES 9(3)497-500, 1988.--In the present study the effect of the newly isolated peptides galanin, calcitonin gene-related peptide (CGRP) and atrial natriuretic peptide (ANP) was examined on spontaneous uterine smooth muscle activity on the rat in vitro. Galanin showed a slight and insignificant stimulatory effect on the amplitude, while both CGRP and ANP were found to be potent inhibitors of the uterine smooth muscle contractions. In connection with the recent demonstration of galanin and CGRP nerves in the genital tract, these pharmacological findings suggest that the peptides may participate in the control of nervous control of uterine muscular activity, although the exact physiological roles remain to be clarified. Neuropeptides Uterine motility Atrial natriuretic peptide (ANP)
Galanin
Calcitonin gene-related peptide (CGRP)
METHOD
A N increasing number of peptides, originally isolated from extragenital sources, have by radioimmunoassay and immunocytochemistry been demonstrated in the genitourinary tract [9]. Recently two newly isolated peptides, galanin and CGRP, have been demonstrated in the female genital tract [2,4]. Galanin is a 29 amino acid peptide, originally isolated from porcine small intestine [12]. In the urogenital system of man and rat galanin immunoreactive nerve fibres are found within smooth muscle and in close relationship to blood vessels [2]. Calcitonin gene-related peptide (CGRP) consists of 37 amino acid residues [11]. This peptide, which has been identified in central and peripheral neural tissues, is a product of m R N A formed by alternative tissue-specific RNA splicing after transcription of the calcitonin gene [1 ]. In the rat genitourinary tract CGRP nerve fibres are associated mainly with blood vessels, non-vascular smooth muscle and epithelial cells [4]. In the uterus CGRP immunoreactive fibres are mainly distributed in the myometrium [4]. Atrial natriuretic peptide (ANP), a 28 amino acid peptide originally isolated from atrial muscle of the heart, has been shown to influence vascular and non-vascular smooth muscle activity [3]. In order to investigate a possible physiological role of the three peptides, galanin, CGRP and A N P in the female genital tract, we examined their effect on the activity of smooth muscle strips from rat uterus.
Animals Female non-pregnant Wistar rats weighing approximately 200 g were employed. Three days before sacrifice the animals received daily injections of oestradiolbenzoate, Ovex ®IM (Loeven, Denmark) (1.25 mg/kg) in order to obtain a spontaneous and rhythmic activity of the uterine musculature.
Tissue Strips On the day of experiment the animals were anaesthetised by intraperitoneal administration of pentobarbitone (1 g/kg). The uterus was immediately removed to ice-cold modified Krebs buffer with the following composition: NaC1 118 mmol/l, KCI 4.4 mmol/1, MgSO4 0.12 mmol/1, KH2PO3 1.2 mmol/l, KC1 2.51 mmol/1, NaHCO3 25 mmol/1 and glucose 11 mmol/1. Tissue strips measuring 1 × 1 × 10 mm were excised and mounted vertically between a horizontal bolt and an isometric force-displacement transducer (Grass FT03C, Grass Instruments, USA) in a 5 ml organ bath containing Krebs buffer thermostatically kept at 37.0°C. The buffer was continuously aerated with a mixture of 95% Oz and 5% CO2. The strips were subjected to tension of approximately 1 g and allowed to equilibrate until spontaneous contractions developed. The contractions were monitored on a linearecorder (Servogor RE 511, Goerz, Austria).
497
498
BEK, O T T E S E N AND F A H R E N K R U G
Galanin A
~
B
5 min.
120-
m
813
~
t
t~ tO
10-10 M
N
1'1
;
10
;
7
t,,-
8
- log conc. M
FIG. 1. (A) Shows the effect of increasing concentrations of galanin on myometrial strips from non-pregnant rat uterus in vitro. Amplitude (dashed line); frequency (solid line). Values (mean+SEM) are percentage change of the spontaneous contractions (n=6). (B) Shows a representative trace. (The short-lived aberrant contraction immediately after application of peptide is an artefact.) ANP
A
B
100I
._Z, .>
5 min.
80o (-
t't .¢- l
60"
E ._c 40-
~' Tt
t 10..8M
O} c'-
.g
20"
3 0
-log cone. M FIG. 2. (A) Shows the effect of increasing concentrations of ANP on myometrial strips from nonpregnant rat uterus in vitro. Amplitude (dashed line); frequency (solid line). Values (mean-+SEM) are percentage change of the spontaneous contractions (n =4). The amplitude was significantly reduced at l0 -9 tool/1 and above. (B) Shows a representative trace.
Experimental Procedure When a regular rhythm of contractions had developed, the dose-response relationship of each peptide was examined by adding ten-fold increasing concentrations to the organ bath. Application of peptide to the strips in a non-relaxed phase induced an initial short-lived and aberrant contraction. The next concentration of peptide was added when activity had returned to basal and at least identical contractions had appeared. The effect of porcine galanin was investigated in a concentration range from 10-11 to 10-7 mol/l, rat CGRP from 10-9 to 10-6 mol/l and rat ANP from 10-11 to 10-~ mol/1. Full dose-response curves for each peptide were established in strips from 4--6 animals. Peptides were synthetic, purchased from Peninsula Laboratories (St. Helens, United Kingdom).
Each tissue strip was its own control. Thus the effect on frequency and amplitude was compared to the frequency and amplitude immediately before peptide application. The effect was expressed as a percent change of the preceding control activity. Results are expressed as means-+-SEM. Statistical analysis was performed using an ANOVA test and in case of significant effect followed by a Newman-Keuls' test. Differences resulting in p-values less than 0.05 were considered significant. RESULTS
Galanin showed a slight and insignificant stimulatory el-
G A L A N I N , CGRP, ANP AND RAT U T E R U S
499
C(3RP
TABLE 1 DOSE-DEPENDENT INCREASEIN THE DURATIONOF INHIBITION OF SPONTANEOUSUTERINEMUSCLE ACTIVITYAFTER APPLICATIONOF ANP AND CGRP (MEAN -+ SEM)
5 min. t
I
Duration of Action (min) Concentration (mol/1) 10-11 10-1° 10-9 10-s 3×10 -8 10-7 3×10 7 10-6
ANP (n=4) 0 2.6 3.2 4.2
-+ 0 -+ 0.9 - 1.2 ± 1.7
4.9 ± 0.2 7.4 ± 1.1
CGRP (n=6)
0 --- 0 1.5 ± 1.0 7.0 ± 1.2 8.5 ± 3.7 16.6 --- 4.9 Total Inhibition
'I 3 x 10-8 M
FIG. 3. Representative trace showing CGRP-induced inhibition of spontaneous activity of myometrial strips from non-pregnant rat uterus. fect on the amplitude of uterine smooth muscle activity, while the frequency was unaffected (Fig. 1). ANP displayed a pronounced inhibitory action on the amplitude of spontaneous uterine contractions. The frequency, however, did not change significantly (Fig. 2). The duration of ANP action until total recovery increased moderately in a dosedependent manner (Table 1). Concentrations of CGRP 10-s mold completely inhibited spontaneous uterine contractions, while concentrations below this value had virtually no effect (Fig. 3). A prominent feature of this peptide was the extensive dose-related increase in duration of inhibition of spontaneous activity (Table 1). DISCUSSION
The present study provides evidence that CGRP, ANP and to a slight degree galanin influence spontaneous smooth muscle activity of the rat uterus. The purpose of pretreating the rats with oestradiolbenzoate was to ensure an estrogen dominated myometrium in all animals and thereby obtaining spontaneous and rhythmic activity of the smooth muscle specimens. Furthermore, this treatment produces identical estrogen levels in all rats, which is desirable since it has been shown that sex steroids are able to influence the susceptibility of smooth musculature to the effect of peptides for example vasoactive intestinal peptide [8]. The modest stimulatory effect of galanin on the amplitude contrasts to the pronounced ability of the peptide to induce contractions of smooth muscle preparations from the gastrointestinal tract and urinary bladder of the rat [12]. While the action and the presence of galanin im-
munoreactivity in the intestinal [5,10] and the urinary tracts [2] suggest that galanin participates in the autonomic nervous control of smooth muscle activity, the role of the peptide in uterine contractility seems questionable. The effect of CGRP was all or none in nature with regard to amplitude of spontaneous muscle activity in the rat uterus, whereas the duration of the inhibitory action increased dose-dependently. The nature of the response to CGRP is not really indicative for a physiological role as efferent neurotransmitter. It has, however, been suggested that CGRP in the genitourinary tract is present in non-myelinated sensory nerve fibres based on the fact that capsaicin pretreatment resulted in a substantial depletion of the CGRP content [4]. ANP was, like CGRP, found to be an inhibitor of uterine smooth muscle activity, and the inhibitory effect of ANP on amplitude was dose-related. At present, ANP has not yet been demonstrated in peripheral neurons and a role for ANP as a local neurotransmitter remains to be clarified. The concentrations of ANP needed to influence uterine smooth muscle activity are above the reported circulating plasma levels. Future studies will probably elucidate the possible role of ANP in the physiological control of the uterine activity. The regulation of uterine smooth muscle activity is complex. Besides the role of the classical adrenergic and cholinergic nerves, VIP and substance P have previously been shown to be inhibitory and stimulatory transmitters, respectively [6,7]. The present findings contribute to make the picture of peptidergic regulation of uterine smooth muscle activity even more complex.
REFERENCES
1. Amara, S. G., V. Jonas, M. G. Rosenfeld, E. S. Ong and R. M. Evans. Alternative RNA processing in calcitonin gene expression generates mRNA encoding different polypeptide products. Nature 298: 240-244, 1982. 2. Bauer, F. E., N. D. Christofides, G. W. Hacker, M. A. Blank, J. M. Polak and S. R. Bloom. Distribution of galanin immunoreactivity in the genitourinary tract of man and rat. Peptides 7: 5-10, 1986. 3. Cantin, M. and J. Genest. The heart and the atrial natriuretic factor. Endocr Rev 6: 107-127, 1986.
4. Ghatei, M. A., P. K. Mulderry, M. A. Blank, J. M. Allen, J. F. B. Morrison, J. M. Polak and S. R. Bloom. Calcitonin generelated peptide in the female rat urogenital tract. Peptides 6: 809-815, 1985. 5. Melander, T., T. Hfkfelt, A. ROkaeus, J. Fahrenkrug, K. Tatemoto and V. Mutt. Distribution of galanin-like immunoreactivity in the gastrointestinal tract of several mammalian species. Cell Tissue Res 239: 253-270, 1980. 6. Ottesen, B. Vasoactive intestinal polypeptide as neurotransmitter in the female genital tract. Am J Obstet Gynecol 147: 208224, 1983.
500 7. Ottesen, B., B. R. Gram and J. Fahrenkrug. Neuropeptides in the female genital tract: Effect on vascular and non-vascular smooth muscle. Peptides 4: 387-392, 1983. 8. Ottesen, B., J.-J. Larsen, P. Staun-Olsen, S. Gammeltoft and J. Fahrenkrug. Influence of pregnancy and sex steroids on concentration, motor effect and receptor binding and VIP in the rabbit female genital tract. Regul Pept 11: 83-92, 1985. 9. Ottesen, B., G. Wagner and J. Fahrenkrug. Peptidergic innervation of sexual organs. In: Handbook of Sexology. Vol 6, edited by J. M. A. Sitsen. Amsterdam: Elsevier, 1988, in press.
BEK, OTTESEN AND FAHRENKRUG 10. R6kaeus, A., T. Melander, T. H6kfelt, J. M. Lundberg, K. Tatemoto, M. Carlquist and V. Mutt. A galanin-like peptide in the central nervous system and intestine of the rat. Neurosci Lett 47: 161-166, 1984. 11. Rosenfeld, M. G., J.-J. Mermord, S. G. Amara, L. W. Swanson, P. E. Sawchenko, J. Rivier, W. W. Vale and R. M. Evans. Production of a novel neuropeptide encoded by the calcitonin gene via tissue specific RNA processing. Nature 304: 12%135, 1983.
12. Tatemoto, K., A. R6kaeus, H. J6rnvall, T. J. McDonald and V. Mutt. Galanin--A novel biologically active peptide from porcine intestine. FEBS Lett 164: 124-128, 1983.