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Isolation and characterization of substance P, substance P 5–11, and substance K from two metastatic ileal carcinoids
Regulatory Peptides, 12 (1985) 185-199 Elsevier
185
RPT 00416
Isolation and characterization of substance P, substance P 5-11, and substance K from two metastatic ileal carcinoids Kevin A. Roth*, George M a k k , O l o f Beck, K y m Faull, Kazuhiko Tatemoto, Christopher J. Evans and Jack D. Barchas Nancy Pritzker Laboratory of Behavioral Neurochemistry, Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, CA 94305, U.S.A. (Received 31 May 1985; revised manuscript received 2 July 1985; accepted for publication 27 July 1985)
Summary Using an antiserum directed at the COOH-terminus of tachykinins, we have examined postmortem tissue from two cases of metastatic ileal carcinoid for the presence of tachykinin-like immunoreactivity. The vast majority of the immunoreactive tachykinin-like material eluted from a Sephadex G-50 column as two peaks at positions corresponding to molecular weights of 1300 and 850. The 1300 dalton peak was resolved by reverse-phase -HPLC into two components which by Edman sequencing, amino acid analysis, and fast atom bombardment (FAB)-mass spectrometry criteria, were identified as substance P and substance K. The 850 dalton peak was also resolved on RP-HPLC into two peaks which were resistant to Edman degradation but from amino acid analysis and FAB-mass spectrometry criteria were identified as pyro-Glu-substance P 5-11 and oxidized pyro-Glu-substance P 5-11. In control experiments substance P 5-11 was converted to pyro-Glu-substance P 5-11 during the extraction procedure. Both tumors also contained a minor immunoreactive peak which eluted from a Sephadex G-50 sizing column at a position corresponding to a molecular weight of 4000 which probably represents neuropeptide K. These results suggest that fl-preprotachykinin is preferentially expressed in carcinoid tumors and that substance K may also play a role in the carcinoid syndrome. substance P; substance P 5-11; substance K; ileal carcinoids; isolation; characterization
* To whom correspondence should be addressed, present address: Department of Pathology, Washington University School of Medicine, St. Louis, MO 63110, U.S.A. 0167-0115/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
186
Introduction
Substance P belongs to a family of peptides, called tachykinins, which possess a common COOH-terminal Phe-X-Gly-Leu-Met-NH2 sequence [1]. Recently, two new tachykinins were discovered in mammalian spinal cord extracts; these compounds are termed neuromedin K (neurokinin fl) and substance K (neurokinin ~t) [2--4] (Fig. 1). Two substance P precursor molecules have been identified in bone striatal mRNA extracts; ~-preprotachykinin contains one copy of substance P, fl-preprotachykinin contains one copy of substance P and one copy of substance K [5,6]. These two preprotachykinins are derived from a single gene by alternative RNA splicing.
Substance P:
Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-MN 2
Substance K:
Hts-Lys-Thr-Asp-Ser-Phe-Val-Gl~-Leu-Met-NH2
Neuromedin K:
Asp-Met-His-Asp-Phe-Phe-Val-Gly-Leu-Met-NH~
Fig. 1. Amino acid sequences ofthemammalian tachykinins:substance P, substance K a n d neuromedin K. The homologous COOH-terminalre~on is underlined.
Substance P-like immunoreactivity has been reported in both tumor extracts and plasma from patients with carcinoid tumors [7-12]. In all cases reported, the vast majority of the substance P-like immunoreactivity found was of similar size and HPLC characteristics as authentic substance P. Using a COOH-terminal directed substance P antisera which had full crossreactivity with known tachykinins, our examination of two metastatic carcinoid tumors revealed the presence of substance K in addition to substance P and substance P 5-11, suggesting the preferential expression of//-preprotachykinin in this type of tumor.
Material and Methods
Radioimmunoassay A COOH-terminal directed substance P antiserum was raised in rabbits against synthetic substance P (Peninsula Labs) linked by a water-soluble carbodiimide to bovine thyroglobulin as described earlier [13]. Substance P antiserum (code R2-2) was used in a dilution of 1:15 000 with 125I.TyrO_substanc e p as tracer and synthetic substance P as standard. The RIA had an IC5o of 200 pM. This antiserum appeared to be COOH-terrninally directed since it had full crossreactivity with substance P, substance P 5-11, substance K, and other tachykinins: physalaemin, eledoisin, and kassinin. There was less than 0.001% crossreactivity with substance P free acid, gastrin releasing peptide and bombesin. Radioimmunoassays were performed as previously described [13] except that separation of bound from free peptide was performed by double antibody immunoprecipitation and a buffer of pH 7.4 was used.
187
Tissue extraction Metastatic carcinoid tumor tissue from two patients with primary ileal carcinoids was obtained at autopsy 8-12 h post mortem and immediately frozen at -70"C. For determination of the total tachykinin-like immunoreactivity concentration in the tumors, a small amount of tumor tissue (100-200 mg) was extracted by sonication in acid/acetone (acetone/water/12 M HCI, 40:6:1) as previously described [14]. Extracts were resuspended in RIA buffer and assayed. For more extensive characterization of the peptides, 15 g of carcinoid liver metastases from case 1 was extracted as previously described [15] except that petroleum ether rather than heptane was used to delipidize the acid/acetone extract and a pH 7.0 RP-HPLC buffer of 4 ml/1 triethylamine and phosphoric acid was used rather than 100 mM disodium phosphate.
Sephadex G-50 column chromatography Evaporated acid/acetone extracts were resuspended in 50% acetic acid and chromatographed on a 120 x 0.9 cm column packed with Sephadex G-50 fine. The column was eluted with 50% acetic acid and 1.5-ml fractions were collected and aliquots evaporated under reduced pressure and assayed.
Reverse-phase high-pressure liquid chromatography Aliquots of the tachykinin-immunoreactive peaks from the Sephadex G-50 sizing column were diluted to 5% acetic acid and approximately 10000 cpm of 125I-Metenkephalin-Arg-Gly-Leu added as an internal standard. Chromatography was performed on an Altex ultrasphere ODS column (250 x 4.6 mm; particle size, 5/~m). Two Altex HPLC pumps and a Beckman gradient-mixing computer were used to generate an acetonitrile gradient of 0-15% in 5 min, followed by a 15-40% gradient in 45 min. The HPLC buffer consisted of 50 mM monosodium phosphate, 8.8 mM phosphoric acid, and 5% methanol, at pH 2.7. Flow rate was 1.25 ml/min and 1-min fractions were collected. Aliquots were evaporated under reduced pressure and assayed.
Peptide sequencing The primary sequence of the purified desalted tachykinin-like peptide was obtained using an Applied Biosystems Gas Phase protein sequencer. Samples were loaded in the presence of polybrene onto a glass fiber filter. The anilinothiazolinone amino acids resulting from the Edman degradation were converted to phenylthiohydantoin amino acids by methanolic HC1 (1 M) and analyzed by RP-HPLC using a Hewlett Packard 1084 HPLC system using a 5 # IBM cyano-column with UV detection (254 rim).
Amino acid analysis Purified desalted peptides were hydrolyzed in 5.7 M HCI/0.5% phenol at 110"C for 24 h in evacuated and sealed tubes. Amino acid compositions were determined with a Biotronik amino acid analyzer LC5000 using ninhydrin colormetric detection.
188
Fast atom bombardment-mass spectrometry Positive ion FAB-mass spectrometry was performed on purified desalted peptides using a double focusing magnetic sector instrument (ZAB SE, VG Analytical Inc., Manchester, U.K.) with xenon as the bombarding species (8 kV, 0.1 mA). Between 10 and 250 pmol of tachykinin-immunoreactive peptide in 1-2/al of 0.1% trifluoroacetic acid was deposited on top of a small droplet (1-2/~1) of monothioglycerol on the FAB probe tip. Samples were analyzed at approximately 1000 nominal resolution (M/AM, 10% valley) with 8 kV accelerating voltage by repetitively scanning a mass window of 150-400 daltons covering the region of the suspected molecular weight of the sample, at a rate of between 2-5 s per scan. Data were collected into the data system and the successive scans from a single sample loading were summed with a multichannel analyzer. In this way, between 20 and 50 scans from a single sample loading, representing a total of 1-2 rain of acquisition time, were accummulated. Mass assignments of the summed spectra were made by comparison with appropriate ions in the spectra of cesium iodide and an equimolar mixture of cesium and rubidium iodides. Positive ion FAB spectra of peptides generally show intense [M + H] ÷ ions and we have relied on this characteristic in the calculation of the molecular weights of each peptide.
Results
Crude acid/acetone extracts of both metastatic ileal carcinoids contained high concentrations of tachykinin-like immunoreactivity. Case 1 had a concentration of 4700 pmol/g tissue and case 2 a concentration of 570 pmol/g tissue. Sephadex G-50 gel filtration resolved both tumor extracts into 2 major peaks of immunoreactivity with approximate molecular weights of 1300 and 850 (Fig. 2). A minor peak of immunoreactivity was also observed in both cases at an approximate molecular weight of 4000. The 2 major peaks of immunoreactivity from both cases were further analyzed by RP-HPLC; the results of this analysis were identical for both cases. RP-HPLC resolved the 1300 dalton peak of immunoreactivity (peak A) into two major peaks (Figs. 3 and 4, top panel). These peaks were labelled I and II. Peak I Peak I eluted at the position of oxidized substance P and substance K. However, after oxidation with 0.3% H202 in 0.1 N HCI the immunoreactive material from Peak I eluted at the retention time of oxidized substance K (Figs. 3 and 4, bottom panel). Analysis of purified peak I showed the amino acid sequence of the first 8 amino acid residues to be His-Lys-Thr-Asp-Ser-Phe-Val-Gly (Table I). Amino acid analysis revealed an amino acid composition of His, Lys, Thr, Asp, Ser, Phe, Val, Gly, Leu, and Met in approximately equimolar concentrations (Table II). Positive ion FAB mass spectroscopy of peak I demonstrated a prominent ion at m/z 1133.4 (Fig. 6A) suggesting a molecular weight for the monoisotopic species of 1132.4. These results indicate that peak I was substance K.
189 I=:1[°
+
10
3510
2340
880
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÷28097
B
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~28159
0
0 C
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8
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B
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20
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4'0
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Fig. 2. Gel-filtration chromatography profiles of tachykinin-like immunoreactivity in tumor extracts of Case 1 (No. 28097) and Case 2 (No. 28159). Arrows indicate exclusion and molecular weight markers.
190 •
400.
b
+28097 Peak A I
II
300"
200"
100 A
=[ cL >, >
0
A
I
a: Ox. Sub. K b: Sub. K and Ox. Sub. P c: Sub. P
o o c3
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Oxidized Peak I
E
_E
a
b
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300"
200
100
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Fig. 3. Top: RP-HPLC of immunoreactive tachykinin peak A from C a ~ 1 (No. 28097). Bottom: RPHPLC of oxidized peak I. Markers: (a) oxidized substance K, (b) substance K and oxidized substance P, (c) substance P.
191 b
4OO ÷28159 Peak A
C
II
300
I
200'
100'
A
__A
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w
i
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a: Ox. Sub. K b: Sub. K and Ox. Sub. P c: Sub. P
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Oxidized Peak I
_s
30o-
200-
100-
0 0
1'0
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Fig. 4. Top: RP-HPLC of immunorcactive tachykinin peak A from Case 2 (No. 28159). Bottom: RPHPLC of oxidized peak I. Markers: (a) oxidized substance K, (b) substance K and oxidized substance P, (c) substance P.
192 TABLE I Amino acid sequence of peaks 1 and I1
Peak I
Peak II
Cycle No.
> PhNCSamino acid
Yield (pmol)
I 2 3 4 5 6 7 8 9
His Lys Thr Asp Ser Phe Val Gly
442 510 476 603 430 119 37 29
1 2 3 4 5 6 7
Arg Pro Lys Pro Gln Gin
350 496 147 145 87 25
Carry over from (n-l) (pmol) 56 116 119 194 110 32 9 11 35 49 14 25 25 0
Approximately 600 pmol (by RIA) of peaks I and II were used in each experiment.
Peak H Peak II eluted from R P - H P L C at the retention time o f substance P. The amino acid sequence o f the first 6 amino acid residues proved to be: Arg-Pro-Lys-ProGln-Gln (Table I). Amino acid analysis revealed an amino acid composition of Arg, Pro, Lys, Glu, Phe, Gly, Leu, and Met in approximate molar concentrations of 1, 2, 1, 2, 2, 1, 1, 1, respectively (Table II). Positive ion FAB mass spectroscopy o f peak II demonstrated a prominent ion at m/z 1347.4 (Fig. 6B) suggesting a molecular weight for the monoisotopic species o f 1346.4. These results indicate that peak II was substance P. R P - H P L C resolved the 850 dalton peak of immunoreactivity (peak B) into two major peaks (Fig. 5), labelled III and IV. Peaks 111 and IV Amino acid analysis of both peaks yielded identical amino acid data; the amino acid composition was: Glu, Phe, Gly, Leu, Met in approximate molar concentrations of 2, 2, 1, 1, 1, respectively (Table II). This corresponds to the amino acid composition of substance P 5-11. FAB-mass spectroscopy data (Fig. 6D and E) suggested molecular weights for the monoisotopic species in peaks III and IV to be 867.5 and 851.3, respectively. Edman degradation of both peaks yielded no sequence data suggesting an N-terminal blockage. An explanation for these results which is consistent with all the data is the presence of N-terminal pyro-Glu-substance P 5-11. The molecular weight of this compound is identical with peak IV and the N-terminal pyr-
193 oglutamic acid g r o u p would explain the resistance to E d m a n degradation. Peak I I I is 16 daltons larger than p e a k IV a n d m a y represent oxidized p y r o - G l u - s u b s t a n c e P 5-11. On R P - H P L C p e a k I I I was f o u n d to coelute with oxidized p y r o - G l u - s u b s t a n c e P 5-11 and p e a k IV co-eluted with non-oxidized p y r o - G l u - s u b s t a n c e P 5-11 (Fig. 5). These findings suggest that p e a k I I I was oxidized p y r o - G l u - s u b s t a n c e P 5-11 and p e a k IV p y r o - G l u - s u b s t a n c e P 5-11. T o determine if substance P 5-11 was cyclized during the extraction procedure, synthetic substance P 5-11 was a d d e d to 400 m g o f guinea pig cerebellum, a tissue without e n d o g e n o u s substance P 5-11, and processed identically to the t u m o r tissue. T h e synthetic substance P 5-11 was purified before use by R P - H P L C to r e m o v e traces o f c o n t a m i n a t i n g p y r o - G l u - s u b s t a n c e P 5-11. T h e tachykinin i m m u n o r e a c t i v e peak f r o m Sephadex G-50 was run on R P - H P L C and assayed. The results showed
TABLE II Amino acid composition of peaks I, II, III and IV Amino acid
Yield (nmol)
Residues per molecule (nearest integer)
Peak I:
His Lys Thr Asp Ser Phe Val Gly Leu Met
0.53 0.53 0.48 0.71 0.64 0.70 0.58 0.63 0.61 0.47
0.90 0.90 0.81 1.20 1.08 1.18 0.98 1.07 1.03 0.80
(1) (1) (1) (1) (1) (1) (1) (1) (1) (I)
Peak II:
Arg Lys Glu Phe Gly Leu Met
0.36 0.28 0.59 0.70 0.31 0.40 0.45
1.05 0.82 1.73 2.05 0.91 1.17 1.49
(1) (1) (2) (2) (1) (1) (1)
Peak III:
Glu Gly Met Leu Phe
1.62 1.07 0.56 1.06 1.73
1.88 1.24 0.64 1.22 2.00
(2) (1) (1) (1) (2)
Peak IV:
Glu Gly Met Leu Phe
1.26 0.87 0.66 0.69 1.15
1.91 1.32 1.00 1.04 1.74
(2) (1) (1) (1) (2)
194 •
400
b
c
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÷28097 Peak B
t
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300
200
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v >t ,m =D om 4,* U
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o
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Fig. 5. Top: RP-HPLC of immunorcactivc tachykinin peak B from Case 1 (No. 28097). Bottom: RPHPLC of immunoreactive tachykinin peak B from Case 2 (No. 28159). Markers: (a) oxidized substance P 5-11, (b) oxidized pyro-Glu-substance P 5-11, (c) substance P 5-11, (d) pyro-Glu-substance P 5-I 1.
195
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UNITS
Fig. 6. Positive ion FAB spectra obtained from approximately 10 pmol of peak I (A), 10 pmol of peak II (B), 100 pmol of peak III (I)) and 250 pmol of peak IV (E). Because insu~cient material was available for repetitive analyses, we cannot provide an estimation of the error associated with the mass measurement for each fraction. However, experience with this technique has indicated an accuracy of the mass measurement which is generaUy better than ± 0.5 daltons. The molecular weights ofthe monoisotopie species of authentic substance K, substance P, oxidized pyro-Glu-substance P 5-11 and pyro-Glu-substance P 5-11 are 1132.5, 1346.7, 867.4, and 851.4, respectively.
196
that nearly 50% of the substance P 5-11 added to the guinea pig cerebellum eluted from RP-HPLC at the retention time of pyro-Glu-substance P 5-11. This result suggests that substance P 5-11 can be pyrolized during the purification procedure. However, in similar control experiments, the addition of synthetic substance P to guinea pig cerebellum did not result in the formation of substance P 5-11.
Discussion
On the basis of HPLC retention times, amino acid analysis, Edman sequencing and molecular weight determination by FAB-mass spectrometry, we have identified substance P, substance P 5-11 and substance K in extracts from two metastatic ileal carcinoids. Substance P-like immunoreactivity has previously been associated with ileal carcinoid tumors and substance P immunoreactive peptides have been found in tumor extracts from patients with the carcinoid syndrome [7-12,16]. Elevated concentrations of substance P immunoreactive material have been found in plasma from patients with carcinoid tumors [8-10]. Characterization of this immunoreactive material by Emson et al. [10] using both N- and C-terminally directed substance P antisera and RP-HPLC suggested that this material represented authentic substance P. Several authors have suggested that substance P immunoreactivity in plasma may prove useful in detecting and localizing carcinoid tumors and may play a role in the clinical manifestations of the carcinoid syndrome [7-10]. In addition to substance P, we identified pyro-Glu-substance P 5-11 and oxidized pyro-Glu-substance P 5-11 in tumor extracts. It is equivocal, however, if substance P 5-11 occurs in tissue in the pyro-Glu form because in control experiments the extraction procedure itself results in the formation of pyro-Glu-substance P 5-11 from substance P 5-11. Hence, we refer to this material simply as substance P 5-11. However, it is clear from control experiments that substance P 5-11 is not formed from substance P by the extraction procedure. Furthermore, previous experiments by several authors on the stability of substance P in post-mortem tissue failed to demonstrate the formation of C-terminal substance P fragments after death, indeed the concentration of C-terminal substance P fragments decreases with time [17,18]. Substance P 5-11 has been reported to occur in low concentrations in the rat brain [18] and this fragment is actively taken up by slices of neural tissue [19]. In most biological systems the COOH-terminal sequence of substance P is required for tachykinin-like activity and in these systems substance P 5-11 is fully active [20]. The conversion of substance P to substance P 5-11 has been demonstrated to occur in vitro in human plasma [21]. Although in the two cases reported here tachykinin-like immunoreactivity in plasma was not examined, a previous report by Conlon et al. [11] of substance P-like immunoreactivity in plasma from a patient with a carcinoid tumor showed that the concentration of C-terminal immunoreactivity was 2-4 times higher than N-terminal immunoreactivity. These results suggest that C-terminal fragments of substance P may circulate in human plasma. Since substance P 5-11 has similar action as substance P in most biological systems, its role in the carcinoid syndrome is probably similar to that proposed for substance P.
197
In this report we demonstrate, for the first time, the prese~ace of substance K in human tumor extracts. The sequence of substance K in humans is identical with the substance K sequence found in other mammalian tissues [3,4]. Using bovine brain messenger RNA, Nawa et al. [5,6] have demonstrated that substance P and substance K are contained in a single gene but that alternative mRNA splicing results in the production of either ~t-preprotachykinin, containing only the substance P sequence, or/~-preprotachykinin, which contains both the substance P and substance K sequences. These two mRNAs appear to be processed in a tissue specific manner, thus in the bovine brain there is a preponderance of 0t-preprotachykinin while in the small intestine there is much more/~-preprotachykinin than ~t-preprotachykinin (6). Carcinoid tumors in the ileum are felt to be derived from neuroendocrine cells normally present in this tissue. Inasmuch as the ratio of ~t- to/~-preprotachykinin in this tissue partially represents the ratio in normal neuroendocrine cells, it seems not unexpected that substance K, which is present in/~-preprotachykinin would be expressed in ileal carcinoids. In several bioassay systems substance K has similar potency as substance P. However, in some systems substance K is much more potent than substance P [22,23]. Recently, Buck et al. [24] reported evidence suggesting that in the rat duodenum and mouse bladder there is a distinct substance K receptor. The presence of this receptor in the small intestine is interesting since substance K is several-fold more potent than substance P in causing contraction of the rat duodenum [23]. The presence of these receptors in humans has not yet been reported but if they are present in the human intestinal tract the effect of elevated substance K concentrations on gastrointestinal function may be important. The recent finding that substance P and substance K stimulate connective tissue cell growth suggests that these compounds may play a role in the marked fibrotic reactions occasionally observed in the carcinoid syndrome [25]. In addition to the immunoreactive peaks at 1300 and 850 daltons both tumor extracts contained a minor peak of immunoreactivity at about 4000 daltons. This peak was not further characterized, however, a likely explanation for this immunoreactivity is the presence of neuropeptide K. Neuropeptide K is a 36 amino acid peptide, corresponding to positions 72-107 in the /~-preprotachykinin precursor, which contains substance K at its COOH-terminus [26]. The possible presence of this compound in carcinoid tumors suggests that the processing of fl-preprotachykinin results in the production of several biologically active peptides. In summary, we have identified several tachykinin-like peptides in two cases of metastatic ileal carcinoid. These peptides included substance P, substance P 5-11, and substance K; the presence of neuropeptide K was also suggested. These tachykinins may play a substantial role in the production of the classic carcinoid syndrome.
Acknowledgements We thank Sue Poage for preparing the manuscript. This work was supported by N I M H program-project grant MH 23861.
198
References 1 Erspamer, V., The tachykinin peptide family, Trends Neurosci,, 4 (1981) 267-269. 2 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. 3 Kimura, S., Okada, M., Sugita, Y., Kamazawa, I. and Munekata, E., Novel neuropeptides, neurokinin-alpha and neurokinin-beta, isolated from porcine spinal-cord, Proc. Jap. Acad. Set. B, 59 (1983) 101-104. 4 Maggio, J.E., Sandberg, B.E.B., Bradley, C.V., Iversen, L.L., Santikam, S., Williams, D.H., Hunter, J.C. and Hanley, M.R., A novel tachykinin in mammalian spinal cord, Irish J. Med. Sci., 152 (S1) (1983) 80. 5 Nawa, H., Hirose, T., Takashima, H., Inayama, S. and Nakanishi, S., Nucleotide sequences of cloned eDNA's for two types of bovine brain substance P precursor, Nature, 306 (1983) 32-36. 6 Nawa, H., Kotani, H. and Nakanishi, S., Tissue-specific generation of two preprotachykinin mRNA's from one gene by alternative RNA splicing, Nature, 312 (1984) 729-734. 7 Skrabanek, P., Cannon, D., Kirrane, J. and Powell, D., Substance P secretion by carcinoid turnouts, Irish J. Med. Sci., 147 (1978) 47-49. 8 Skrabanek, P., Dervan, P., Cannon, D. and Powell, D., Substance P in ovarian carcinoid, J. Clin. Pathol., 33 (1980) 160-162. 9 Strodel, W.E., Vinik, A.I., Jaffe, B.M., Eckhauser, F.E. and Thompson, N.W., Substance P in the localization of a carcinoid tumor, J. Surg. Oncol., 27 (1984) 106-111. 10 Emson, P.C., Gilbert, R.F.T., Martensson, H. and Nobin, A., Elevated concentrations of substance P and 5-HT in plasma in patients with carcinoid tumors, Cancer, 54 (1984) 715-718. 11 Conlon, J.M., Richter, G. and Creutzfeldt, W., Metabolism of substance P in the circulation of a patient with carcinoid syndrome, Dig. Dis. Sci., 29 (Aug. Suppl.), (1984) 18S. 12 Schafer, G., Schmidt, W.E., Weber, M. and Conlon, J.M., Characterization and biosynthesis of substance P-related peptides in a carcinoid tumor, Dig. Dis. Sci., 29 (Aug. Suppl.) (1984) 73S. 13 Weber, E., Evans, C.J., Chang, J-K. and Barchas, J.D., Antibodies specific for ~-N-acetyl ]~-endorphins: radioimmunoassays and detection of acetylated//-endorphins in pituitary extracts, J. Neurochem., 38 (1982) 436-447. 14 Roth, K.A., Evans, C.J., Weber, E., Barchas, J.D., Bostwick, D.G. and Bensch, K.G., Gastrin-releasing peptide-related peptides in a human malignant lung carcinoid tumor, Cancer Res., 43 (1983) 5411-5415. 15 Weber, E., Esch, F.S., Bohlen, P., Paterson, S., Corbett, A.D., MeKnight, A.T., Kosterlitz, H.W., Barchas, J.D. and Evans, C.J., Metorphamide: Isolation, structure, and biologic activity of an amidated opioid octapeptide from bovine brain, Proc. Natl. Acad. Sci. USA, 80 (1983) 7362-7366. 16 Hakanson, R., Bengmark, S., Brodin, E., Ingemansson, S., Larsson, L.-I., Nilsson, G. and Sunder, F., Substance P-like immtmoreactivity in intestinal carcinoid tumors. In U.S. yon Euler and B. Perrow (Eds.), Substance P, Raven Press, New York, 1977, pp. 55-58. 17 McGregor, G.P. and Bloom, S.R., Radioimmunoassay of substance P and its stability in tissue, Life Sci., 32 (1983) 655-662. 18 Sakurada, T., Le Greves, P., Stewart, J. and Terenius, L., Measurement of substance P metabolites in rat CNS, J. Neurochem., 44 (1985) 718-722. 19 Nakata, Y., Kusaka, Y., Yajima, H. and Segawa, T., Active uptake of substance P carboxyterminal heptapeptide (5-11) into rat brain and rabbit spinal cord slices, J. Neurochem., 37 (1981) 1529-1534. 20 Pernow, B., Substance P, Pharmacol. Rev., 35 (1983) 85-141. 21 Conlon, J.M. and Sheehan, L., Conversion of substance P to C-terminal fragments in human plasma, Regul. Peptides, 7 (1983) 335-345. 22 Hunter, J.C. and Maggio, J.E., Pharmacological characterization of a novel tachykinin isolated from mammalian spinal cord, Eur. J. Pharmacol., 97 (1984) 159-160. 23 Nawa, H., Doteuchi, M., Igano, K., Inouye, K. and Nakanishi, S., Substance K: a novel mammalian tachykinin that differs from substance P in its pharmacological profile, Life Sci., 34 (1984) 1153-1160. 24 Buck, S. H., Burcher, E., Shults, C.W., Lovenberg, W. and O'Donohue, T.L., Novel pharmacology of substance K-binding sites: a third type of tachykinin receptor, Science, 226 (1984) 987-989.
199 25 Nilsson, J., von Euler, A.M. and Dalsgaard, C.-J., Stimulation of connective tissue cell growth by substance P and substance K, Nature, 315 (1985) 61-63. 26 Tatemoto, K,, Lundberg, J.M., Jornvall, H. and Mutt, V., Neuropeptide K: Isolation, structure and biological activities of a novel brain tachykinin, Biochem. Biophys. Res. Commun., 128 (1985) 947953.
185
RPT 00416
Isolation and characterization of substance P, substance P 5-11, and substance K from two metastatic ileal carcinoids Kevin A. Roth*, George M a k k , O l o f Beck, K y m Faull, Kazuhiko Tatemoto, Christopher J. Evans and Jack D. Barchas Nancy Pritzker Laboratory of Behavioral Neurochemistry, Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, CA 94305, U.S.A. (Received 31 May 1985; revised manuscript received 2 July 1985; accepted for publication 27 July 1985)
Summary Using an antiserum directed at the COOH-terminus of tachykinins, we have examined postmortem tissue from two cases of metastatic ileal carcinoid for the presence of tachykinin-like immunoreactivity. The vast majority of the immunoreactive tachykinin-like material eluted from a Sephadex G-50 column as two peaks at positions corresponding to molecular weights of 1300 and 850. The 1300 dalton peak was resolved by reverse-phase -HPLC into two components which by Edman sequencing, amino acid analysis, and fast atom bombardment (FAB)-mass spectrometry criteria, were identified as substance P and substance K. The 850 dalton peak was also resolved on RP-HPLC into two peaks which were resistant to Edman degradation but from amino acid analysis and FAB-mass spectrometry criteria were identified as pyro-Glu-substance P 5-11 and oxidized pyro-Glu-substance P 5-11. In control experiments substance P 5-11 was converted to pyro-Glu-substance P 5-11 during the extraction procedure. Both tumors also contained a minor immunoreactive peak which eluted from a Sephadex G-50 sizing column at a position corresponding to a molecular weight of 4000 which probably represents neuropeptide K. These results suggest that fl-preprotachykinin is preferentially expressed in carcinoid tumors and that substance K may also play a role in the carcinoid syndrome. substance P; substance P 5-11; substance K; ileal carcinoids; isolation; characterization
* To whom correspondence should be addressed, present address: Department of Pathology, Washington University School of Medicine, St. Louis, MO 63110, U.S.A. 0167-0115/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
186
Introduction
Substance P belongs to a family of peptides, called tachykinins, which possess a common COOH-terminal Phe-X-Gly-Leu-Met-NH2 sequence [1]. Recently, two new tachykinins were discovered in mammalian spinal cord extracts; these compounds are termed neuromedin K (neurokinin fl) and substance K (neurokinin ~t) [2--4] (Fig. 1). Two substance P precursor molecules have been identified in bone striatal mRNA extracts; ~-preprotachykinin contains one copy of substance P, fl-preprotachykinin contains one copy of substance P and one copy of substance K [5,6]. These two preprotachykinins are derived from a single gene by alternative RNA splicing.
Substance P:
Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-MN 2
Substance K:
Hts-Lys-Thr-Asp-Ser-Phe-Val-Gl~-Leu-Met-NH2
Neuromedin K:
Asp-Met-His-Asp-Phe-Phe-Val-Gly-Leu-Met-NH~
Fig. 1. Amino acid sequences ofthemammalian tachykinins:substance P, substance K a n d neuromedin K. The homologous COOH-terminalre~on is underlined.
Substance P-like immunoreactivity has been reported in both tumor extracts and plasma from patients with carcinoid tumors [7-12]. In all cases reported, the vast majority of the substance P-like immunoreactivity found was of similar size and HPLC characteristics as authentic substance P. Using a COOH-terminal directed substance P antisera which had full crossreactivity with known tachykinins, our examination of two metastatic carcinoid tumors revealed the presence of substance K in addition to substance P and substance P 5-11, suggesting the preferential expression of//-preprotachykinin in this type of tumor.
Material and Methods
Radioimmunoassay A COOH-terminal directed substance P antiserum was raised in rabbits against synthetic substance P (Peninsula Labs) linked by a water-soluble carbodiimide to bovine thyroglobulin as described earlier [13]. Substance P antiserum (code R2-2) was used in a dilution of 1:15 000 with 125I.TyrO_substanc e p as tracer and synthetic substance P as standard. The RIA had an IC5o of 200 pM. This antiserum appeared to be COOH-terrninally directed since it had full crossreactivity with substance P, substance P 5-11, substance K, and other tachykinins: physalaemin, eledoisin, and kassinin. There was less than 0.001% crossreactivity with substance P free acid, gastrin releasing peptide and bombesin. Radioimmunoassays were performed as previously described [13] except that separation of bound from free peptide was performed by double antibody immunoprecipitation and a buffer of pH 7.4 was used.
187
Tissue extraction Metastatic carcinoid tumor tissue from two patients with primary ileal carcinoids was obtained at autopsy 8-12 h post mortem and immediately frozen at -70"C. For determination of the total tachykinin-like immunoreactivity concentration in the tumors, a small amount of tumor tissue (100-200 mg) was extracted by sonication in acid/acetone (acetone/water/12 M HCI, 40:6:1) as previously described [14]. Extracts were resuspended in RIA buffer and assayed. For more extensive characterization of the peptides, 15 g of carcinoid liver metastases from case 1 was extracted as previously described [15] except that petroleum ether rather than heptane was used to delipidize the acid/acetone extract and a pH 7.0 RP-HPLC buffer of 4 ml/1 triethylamine and phosphoric acid was used rather than 100 mM disodium phosphate.
Sephadex G-50 column chromatography Evaporated acid/acetone extracts were resuspended in 50% acetic acid and chromatographed on a 120 x 0.9 cm column packed with Sephadex G-50 fine. The column was eluted with 50% acetic acid and 1.5-ml fractions were collected and aliquots evaporated under reduced pressure and assayed.
Reverse-phase high-pressure liquid chromatography Aliquots of the tachykinin-immunoreactive peaks from the Sephadex G-50 sizing column were diluted to 5% acetic acid and approximately 10000 cpm of 125I-Metenkephalin-Arg-Gly-Leu added as an internal standard. Chromatography was performed on an Altex ultrasphere ODS column (250 x 4.6 mm; particle size, 5/~m). Two Altex HPLC pumps and a Beckman gradient-mixing computer were used to generate an acetonitrile gradient of 0-15% in 5 min, followed by a 15-40% gradient in 45 min. The HPLC buffer consisted of 50 mM monosodium phosphate, 8.8 mM phosphoric acid, and 5% methanol, at pH 2.7. Flow rate was 1.25 ml/min and 1-min fractions were collected. Aliquots were evaporated under reduced pressure and assayed.
Peptide sequencing The primary sequence of the purified desalted tachykinin-like peptide was obtained using an Applied Biosystems Gas Phase protein sequencer. Samples were loaded in the presence of polybrene onto a glass fiber filter. The anilinothiazolinone amino acids resulting from the Edman degradation were converted to phenylthiohydantoin amino acids by methanolic HC1 (1 M) and analyzed by RP-HPLC using a Hewlett Packard 1084 HPLC system using a 5 # IBM cyano-column with UV detection (254 rim).
Amino acid analysis Purified desalted peptides were hydrolyzed in 5.7 M HCI/0.5% phenol at 110"C for 24 h in evacuated and sealed tubes. Amino acid compositions were determined with a Biotronik amino acid analyzer LC5000 using ninhydrin colormetric detection.
188
Fast atom bombardment-mass spectrometry Positive ion FAB-mass spectrometry was performed on purified desalted peptides using a double focusing magnetic sector instrument (ZAB SE, VG Analytical Inc., Manchester, U.K.) with xenon as the bombarding species (8 kV, 0.1 mA). Between 10 and 250 pmol of tachykinin-immunoreactive peptide in 1-2/al of 0.1% trifluoroacetic acid was deposited on top of a small droplet (1-2/~1) of monothioglycerol on the FAB probe tip. Samples were analyzed at approximately 1000 nominal resolution (M/AM, 10% valley) with 8 kV accelerating voltage by repetitively scanning a mass window of 150-400 daltons covering the region of the suspected molecular weight of the sample, at a rate of between 2-5 s per scan. Data were collected into the data system and the successive scans from a single sample loading were summed with a multichannel analyzer. In this way, between 20 and 50 scans from a single sample loading, representing a total of 1-2 rain of acquisition time, were accummulated. Mass assignments of the summed spectra were made by comparison with appropriate ions in the spectra of cesium iodide and an equimolar mixture of cesium and rubidium iodides. Positive ion FAB spectra of peptides generally show intense [M + H] ÷ ions and we have relied on this characteristic in the calculation of the molecular weights of each peptide.
Results
Crude acid/acetone extracts of both metastatic ileal carcinoids contained high concentrations of tachykinin-like immunoreactivity. Case 1 had a concentration of 4700 pmol/g tissue and case 2 a concentration of 570 pmol/g tissue. Sephadex G-50 gel filtration resolved both tumor extracts into 2 major peaks of immunoreactivity with approximate molecular weights of 1300 and 850 (Fig. 2). A minor peak of immunoreactivity was also observed in both cases at an approximate molecular weight of 4000. The 2 major peaks of immunoreactivity from both cases were further analyzed by RP-HPLC; the results of this analysis were identical for both cases. RP-HPLC resolved the 1300 dalton peak of immunoreactivity (peak A) into two major peaks (Figs. 3 and 4, top panel). These peaks were labelled I and II. Peak I Peak I eluted at the position of oxidized substance P and substance K. However, after oxidation with 0.3% H202 in 0.1 N HCI the immunoreactive material from Peak I eluted at the retention time of oxidized substance K (Figs. 3 and 4, bottom panel). Analysis of purified peak I showed the amino acid sequence of the first 8 amino acid residues to be His-Lys-Thr-Asp-Ser-Phe-Val-Gly (Table I). Amino acid analysis revealed an amino acid composition of His, Lys, Thr, Asp, Ser, Phe, Val, Gly, Leu, and Met in approximately equimolar concentrations (Table II). Positive ion FAB mass spectroscopy of peak I demonstrated a prominent ion at m/z 1133.4 (Fig. 6A) suggesting a molecular weight for the monoisotopic species of 1132.4. These results indicate that peak I was substance K.
189 I=:1[°
+
10
3510
2340
880
A
÷28097
B
.o
4-+ (J
_e 0
i
2'
o
E
0
+
+
lO
+
+
~28159
0
0 C
E E
8
A
B
C
0
20
3'0
4'0
5'o
a'o
fractions
Fig. 2. Gel-filtration chromatography profiles of tachykinin-like immunoreactivity in tumor extracts of Case 1 (No. 28097) and Case 2 (No. 28159). Arrows indicate exclusion and molecular weight markers.
190 •
400.
b
+28097 Peak A I
II
300"
200"
100 A
=[ cL >, >
0
A
I
a: Ox. Sub. K b: Sub. K and Ox. Sub. P c: Sub. P
o o c3
i
400.
Oxidized Peak I
E
_E
a
b
2o
s'o
300"
200
100
o
o
1~
A
4"o
s~
fractions
Fig. 3. Top: RP-HPLC of immunoreactive tachykinin peak A from C a ~ 1 (No. 28097). Bottom: RPHPLC of oxidized peak I. Markers: (a) oxidized substance K, (b) substance K and oxidized substance P, (c) substance P.
191 b
4OO ÷28159 Peak A
C
II
300
I
200'
100'
A
__A
O. 0
w
i
!
a: Ox. Sub. K b: Sub. K and Ox. Sub. P c: Sub. P
> 4.,* (,) (9
P o 400¢: :l E
Oxidized Peak I
_s
30o-
200-
100-
0 0
1'0
2'o
3'0
4'0
;0
fractions
Fig. 4. Top: RP-HPLC of immunorcactive tachykinin peak A from Case 2 (No. 28159). Bottom: RPHPLC of oxidized peak I. Markers: (a) oxidized substance K, (b) substance K and oxidized substance P, (c) substance P.
192 TABLE I Amino acid sequence of peaks 1 and I1
Peak I
Peak II
Cycle No.
> PhNCSamino acid
Yield (pmol)
I 2 3 4 5 6 7 8 9
His Lys Thr Asp Ser Phe Val Gly
442 510 476 603 430 119 37 29
1 2 3 4 5 6 7
Arg Pro Lys Pro Gln Gin
350 496 147 145 87 25
Carry over from (n-l) (pmol) 56 116 119 194 110 32 9 11 35 49 14 25 25 0
Approximately 600 pmol (by RIA) of peaks I and II were used in each experiment.
Peak H Peak II eluted from R P - H P L C at the retention time o f substance P. The amino acid sequence o f the first 6 amino acid residues proved to be: Arg-Pro-Lys-ProGln-Gln (Table I). Amino acid analysis revealed an amino acid composition of Arg, Pro, Lys, Glu, Phe, Gly, Leu, and Met in approximate molar concentrations of 1, 2, 1, 2, 2, 1, 1, 1, respectively (Table II). Positive ion FAB mass spectroscopy o f peak II demonstrated a prominent ion at m/z 1347.4 (Fig. 6B) suggesting a molecular weight for the monoisotopic species o f 1346.4. These results indicate that peak II was substance P. R P - H P L C resolved the 850 dalton peak of immunoreactivity (peak B) into two major peaks (Fig. 5), labelled III and IV. Peaks 111 and IV Amino acid analysis of both peaks yielded identical amino acid data; the amino acid composition was: Glu, Phe, Gly, Leu, Met in approximate molar concentrations of 2, 2, 1, 1, 1, respectively (Table II). This corresponds to the amino acid composition of substance P 5-11. FAB-mass spectroscopy data (Fig. 6D and E) suggested molecular weights for the monoisotopic species in peaks III and IV to be 867.5 and 851.3, respectively. Edman degradation of both peaks yielded no sequence data suggesting an N-terminal blockage. An explanation for these results which is consistent with all the data is the presence of N-terminal pyro-Glu-substance P 5-11. The molecular weight of this compound is identical with peak IV and the N-terminal pyr-
193 oglutamic acid g r o u p would explain the resistance to E d m a n degradation. Peak I I I is 16 daltons larger than p e a k IV a n d m a y represent oxidized p y r o - G l u - s u b s t a n c e P 5-11. On R P - H P L C p e a k I I I was f o u n d to coelute with oxidized p y r o - G l u - s u b s t a n c e P 5-11 and p e a k IV co-eluted with non-oxidized p y r o - G l u - s u b s t a n c e P 5-11 (Fig. 5). These findings suggest that p e a k I I I was oxidized p y r o - G l u - s u b s t a n c e P 5-11 and p e a k IV p y r o - G l u - s u b s t a n c e P 5-11. T o determine if substance P 5-11 was cyclized during the extraction procedure, synthetic substance P 5-11 was a d d e d to 400 m g o f guinea pig cerebellum, a tissue without e n d o g e n o u s substance P 5-11, and processed identically to the t u m o r tissue. T h e synthetic substance P 5-11 was purified before use by R P - H P L C to r e m o v e traces o f c o n t a m i n a t i n g p y r o - G l u - s u b s t a n c e P 5-11. T h e tachykinin i m m u n o r e a c t i v e peak f r o m Sephadex G-50 was run on R P - H P L C and assayed. The results showed
TABLE II Amino acid composition of peaks I, II, III and IV Amino acid
Yield (nmol)
Residues per molecule (nearest integer)
Peak I:
His Lys Thr Asp Ser Phe Val Gly Leu Met
0.53 0.53 0.48 0.71 0.64 0.70 0.58 0.63 0.61 0.47
0.90 0.90 0.81 1.20 1.08 1.18 0.98 1.07 1.03 0.80
(1) (1) (1) (1) (1) (1) (1) (1) (1) (I)
Peak II:
Arg Lys Glu Phe Gly Leu Met
0.36 0.28 0.59 0.70 0.31 0.40 0.45
1.05 0.82 1.73 2.05 0.91 1.17 1.49
(1) (1) (2) (2) (1) (1) (1)
Peak III:
Glu Gly Met Leu Phe
1.62 1.07 0.56 1.06 1.73
1.88 1.24 0.64 1.22 2.00
(2) (1) (1) (1) (2)
Peak IV:
Glu Gly Met Leu Phe
1.26 0.87 0.66 0.69 1.15
1.91 1.32 1.00 1.04 1.74
(2) (1) (1) (1) (2)
194 •
400
b
c
t ~
÷28097 Peak B
t
d
i IV <:>
300
200
III 100
v >t ,m =D om 4,* U
0 a: Ox. Sub. P(6-11) b: Ox. p-Ilu"lub. P(8-11) c: Sub. P(B-11) d: p"lllu-Sub. P(8-1 1)
o
•
400" ~'28159 E Peak B
b
t ~
c
d
t
t
_s
IV <:7 300-
200.
III 100.
0
lo
io
3'0
4o
5'0
e'o
fractions
Fig. 5. Top: RP-HPLC of immunorcactivc tachykinin peak B from Case 1 (No. 28097). Bottom: RPHPLC of immunoreactive tachykinin peak B from Case 2 (No. 28159). Markers: (a) oxidized substance P 5-11, (b) oxidized pyro-Glu-substance P 5-11, (c) substance P 5-11, (d) pyro-Glu-substance P 5-I 1.
195
0"421A
PEAK'I
.
.
.
.
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.
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CESIUM RUBIDIUM IODIDE Cs4~Rb~IS
\
i
1335.97
0
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~
"~ ';~'-'-
--~
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tZ 3:
2.9
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i 868,5 I
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7.9
1852.3
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F CESIUM I~UBIOIUMIODIDE
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" IN
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UNITS
Fig. 6. Positive ion FAB spectra obtained from approximately 10 pmol of peak I (A), 10 pmol of peak II (B), 100 pmol of peak III (I)) and 250 pmol of peak IV (E). Because insu~cient material was available for repetitive analyses, we cannot provide an estimation of the error associated with the mass measurement for each fraction. However, experience with this technique has indicated an accuracy of the mass measurement which is generaUy better than ± 0.5 daltons. The molecular weights ofthe monoisotopie species of authentic substance K, substance P, oxidized pyro-Glu-substance P 5-11 and pyro-Glu-substance P 5-11 are 1132.5, 1346.7, 867.4, and 851.4, respectively.
196
that nearly 50% of the substance P 5-11 added to the guinea pig cerebellum eluted from RP-HPLC at the retention time of pyro-Glu-substance P 5-11. This result suggests that substance P 5-11 can be pyrolized during the purification procedure. However, in similar control experiments, the addition of synthetic substance P to guinea pig cerebellum did not result in the formation of substance P 5-11.
Discussion
On the basis of HPLC retention times, amino acid analysis, Edman sequencing and molecular weight determination by FAB-mass spectrometry, we have identified substance P, substance P 5-11 and substance K in extracts from two metastatic ileal carcinoids. Substance P-like immunoreactivity has previously been associated with ileal carcinoid tumors and substance P immunoreactive peptides have been found in tumor extracts from patients with the carcinoid syndrome [7-12,16]. Elevated concentrations of substance P immunoreactive material have been found in plasma from patients with carcinoid tumors [8-10]. Characterization of this immunoreactive material by Emson et al. [10] using both N- and C-terminally directed substance P antisera and RP-HPLC suggested that this material represented authentic substance P. Several authors have suggested that substance P immunoreactivity in plasma may prove useful in detecting and localizing carcinoid tumors and may play a role in the clinical manifestations of the carcinoid syndrome [7-10]. In addition to substance P, we identified pyro-Glu-substance P 5-11 and oxidized pyro-Glu-substance P 5-11 in tumor extracts. It is equivocal, however, if substance P 5-11 occurs in tissue in the pyro-Glu form because in control experiments the extraction procedure itself results in the formation of pyro-Glu-substance P 5-11 from substance P 5-11. Hence, we refer to this material simply as substance P 5-11. However, it is clear from control experiments that substance P 5-11 is not formed from substance P by the extraction procedure. Furthermore, previous experiments by several authors on the stability of substance P in post-mortem tissue failed to demonstrate the formation of C-terminal substance P fragments after death, indeed the concentration of C-terminal substance P fragments decreases with time [17,18]. Substance P 5-11 has been reported to occur in low concentrations in the rat brain [18] and this fragment is actively taken up by slices of neural tissue [19]. In most biological systems the COOH-terminal sequence of substance P is required for tachykinin-like activity and in these systems substance P 5-11 is fully active [20]. The conversion of substance P to substance P 5-11 has been demonstrated to occur in vitro in human plasma [21]. Although in the two cases reported here tachykinin-like immunoreactivity in plasma was not examined, a previous report by Conlon et al. [11] of substance P-like immunoreactivity in plasma from a patient with a carcinoid tumor showed that the concentration of C-terminal immunoreactivity was 2-4 times higher than N-terminal immunoreactivity. These results suggest that C-terminal fragments of substance P may circulate in human plasma. Since substance P 5-11 has similar action as substance P in most biological systems, its role in the carcinoid syndrome is probably similar to that proposed for substance P.
197
In this report we demonstrate, for the first time, the prese~ace of substance K in human tumor extracts. The sequence of substance K in humans is identical with the substance K sequence found in other mammalian tissues [3,4]. Using bovine brain messenger RNA, Nawa et al. [5,6] have demonstrated that substance P and substance K are contained in a single gene but that alternative mRNA splicing results in the production of either ~t-preprotachykinin, containing only the substance P sequence, or/~-preprotachykinin, which contains both the substance P and substance K sequences. These two mRNAs appear to be processed in a tissue specific manner, thus in the bovine brain there is a preponderance of 0t-preprotachykinin while in the small intestine there is much more/~-preprotachykinin than ~t-preprotachykinin (6). Carcinoid tumors in the ileum are felt to be derived from neuroendocrine cells normally present in this tissue. Inasmuch as the ratio of ~t- to/~-preprotachykinin in this tissue partially represents the ratio in normal neuroendocrine cells, it seems not unexpected that substance K, which is present in/~-preprotachykinin would be expressed in ileal carcinoids. In several bioassay systems substance K has similar potency as substance P. However, in some systems substance K is much more potent than substance P [22,23]. Recently, Buck et al. [24] reported evidence suggesting that in the rat duodenum and mouse bladder there is a distinct substance K receptor. The presence of this receptor in the small intestine is interesting since substance K is several-fold more potent than substance P in causing contraction of the rat duodenum [23]. The presence of these receptors in humans has not yet been reported but if they are present in the human intestinal tract the effect of elevated substance K concentrations on gastrointestinal function may be important. The recent finding that substance P and substance K stimulate connective tissue cell growth suggests that these compounds may play a role in the marked fibrotic reactions occasionally observed in the carcinoid syndrome [25]. In addition to the immunoreactive peaks at 1300 and 850 daltons both tumor extracts contained a minor peak of immunoreactivity at about 4000 daltons. This peak was not further characterized, however, a likely explanation for this immunoreactivity is the presence of neuropeptide K. Neuropeptide K is a 36 amino acid peptide, corresponding to positions 72-107 in the /~-preprotachykinin precursor, which contains substance K at its COOH-terminus [26]. The possible presence of this compound in carcinoid tumors suggests that the processing of fl-preprotachykinin results in the production of several biologically active peptides. In summary, we have identified several tachykinin-like peptides in two cases of metastatic ileal carcinoid. These peptides included substance P, substance P 5-11, and substance K; the presence of neuropeptide K was also suggested. These tachykinins may play a substantial role in the production of the classic carcinoid syndrome.
Acknowledgements We thank Sue Poage for preparing the manuscript. This work was supported by N I M H program-project grant MH 23861.
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References 1 Erspamer, V., The tachykinin peptide family, Trends Neurosci,, 4 (1981) 267-269. 2 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. 3 Kimura, S., Okada, M., Sugita, Y., Kamazawa, I. and Munekata, E., Novel neuropeptides, neurokinin-alpha and neurokinin-beta, isolated from porcine spinal-cord, Proc. Jap. Acad. Set. B, 59 (1983) 101-104. 4 Maggio, J.E., Sandberg, B.E.B., Bradley, C.V., Iversen, L.L., Santikam, S., Williams, D.H., Hunter, J.C. and Hanley, M.R., A novel tachykinin in mammalian spinal cord, Irish J. Med. Sci., 152 (S1) (1983) 80. 5 Nawa, H., Hirose, T., Takashima, H., Inayama, S. and Nakanishi, S., Nucleotide sequences of cloned eDNA's for two types of bovine brain substance P precursor, Nature, 306 (1983) 32-36. 6 Nawa, H., Kotani, H. and Nakanishi, S., Tissue-specific generation of two preprotachykinin mRNA's from one gene by alternative RNA splicing, Nature, 312 (1984) 729-734. 7 Skrabanek, P., Cannon, D., Kirrane, J. and Powell, D., Substance P secretion by carcinoid turnouts, Irish J. Med. Sci., 147 (1978) 47-49. 8 Skrabanek, P., Dervan, P., Cannon, D. and Powell, D., Substance P in ovarian carcinoid, J. Clin. Pathol., 33 (1980) 160-162. 9 Strodel, W.E., Vinik, A.I., Jaffe, B.M., Eckhauser, F.E. and Thompson, N.W., Substance P in the localization of a carcinoid tumor, J. Surg. Oncol., 27 (1984) 106-111. 10 Emson, P.C., Gilbert, R.F.T., Martensson, H. and Nobin, A., Elevated concentrations of substance P and 5-HT in plasma in patients with carcinoid tumors, Cancer, 54 (1984) 715-718. 11 Conlon, J.M., Richter, G. and Creutzfeldt, W., Metabolism of substance P in the circulation of a patient with carcinoid syndrome, Dig. Dis. Sci., 29 (Aug. Suppl.), (1984) 18S. 12 Schafer, G., Schmidt, W.E., Weber, M. and Conlon, J.M., Characterization and biosynthesis of substance P-related peptides in a carcinoid tumor, Dig. Dis. Sci., 29 (Aug. Suppl.) (1984) 73S. 13 Weber, E., Evans, C.J., Chang, J-K. and Barchas, J.D., Antibodies specific for ~-N-acetyl ]~-endorphins: radioimmunoassays and detection of acetylated//-endorphins in pituitary extracts, J. Neurochem., 38 (1982) 436-447. 14 Roth, K.A., Evans, C.J., Weber, E., Barchas, J.D., Bostwick, D.G. and Bensch, K.G., Gastrin-releasing peptide-related peptides in a human malignant lung carcinoid tumor, Cancer Res., 43 (1983) 5411-5415. 15 Weber, E., Esch, F.S., Bohlen, P., Paterson, S., Corbett, A.D., MeKnight, A.T., Kosterlitz, H.W., Barchas, J.D. and Evans, C.J., Metorphamide: Isolation, structure, and biologic activity of an amidated opioid octapeptide from bovine brain, Proc. Natl. Acad. Sci. USA, 80 (1983) 7362-7366. 16 Hakanson, R., Bengmark, S., Brodin, E., Ingemansson, S., Larsson, L.-I., Nilsson, G. and Sunder, F., Substance P-like immtmoreactivity in intestinal carcinoid tumors. In U.S. yon Euler and B. Perrow (Eds.), Substance P, Raven Press, New York, 1977, pp. 55-58. 17 McGregor, G.P. and Bloom, S.R., Radioimmunoassay of substance P and its stability in tissue, Life Sci., 32 (1983) 655-662. 18 Sakurada, T., Le Greves, P., Stewart, J. and Terenius, L., Measurement of substance P metabolites in rat CNS, J. Neurochem., 44 (1985) 718-722. 19 Nakata, Y., Kusaka, Y., Yajima, H. and Segawa, T., Active uptake of substance P carboxyterminal heptapeptide (5-11) into rat brain and rabbit spinal cord slices, J. Neurochem., 37 (1981) 1529-1534. 20 Pernow, B., Substance P, Pharmacol. Rev., 35 (1983) 85-141. 21 Conlon, J.M. and Sheehan, L., Conversion of substance P to C-terminal fragments in human plasma, Regul. Peptides, 7 (1983) 335-345. 22 Hunter, J.C. and Maggio, J.E., Pharmacological characterization of a novel tachykinin isolated from mammalian spinal cord, Eur. J. Pharmacol., 97 (1984) 159-160. 23 Nawa, H., Doteuchi, M., Igano, K., Inouye, K. and Nakanishi, S., Substance K: a novel mammalian tachykinin that differs from substance P in its pharmacological profile, Life Sci., 34 (1984) 1153-1160. 24 Buck, S. H., Burcher, E., Shults, C.W., Lovenberg, W. and O'Donohue, T.L., Novel pharmacology of substance K-binding sites: a third type of tachykinin receptor, Science, 226 (1984) 987-989.
199 25 Nilsson, J., von Euler, A.M. and Dalsgaard, C.-J., Stimulation of connective tissue cell growth by substance P and substance K, Nature, 315 (1985) 61-63. 26 Tatemoto, K,, Lundberg, J.M., Jornvall, H. and Mutt, V., Neuropeptide K: Isolation, structure and biological activities of a novel brain tachykinin, Biochem. Biophys. Res. Commun., 128 (1985) 947953.