A novel radioimmunoassay for neuromedin K. I. Absence of neuromedin K-like immunoreactivity in guinea pig ileum and urinary bladder. II. Heterogeneity of tachykinins in guinea pig tissues

Regulatory Peptides, 26 (1989) 93-105

93

Elsevier REGPEP 00833

A novel radioimmunoassay for neuromedin K. I. Absence of neuromedin K-like immunoreactivity in guinea pig ileum and urinary bladder. II. Heterogeneity of tachykinins in guinea pig tissues Heng-Phon Too, Jose L. Cordova and John E. Maggio Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115 (U.S.A.)

(Received 28 February 1989; revisedversionreceived and accepted22 May 1989) K e y words: Tachykinin; Neurokinin; Substance P; Substance K; Neuromedin K;

Radioimmunoassay

Summary A novel and highly specific radioimmunoassay for the tachykinin peptide neuromedin K (NMK, also known as neurokinin fl, neurokinin B) has been developed and used to determine the distribution of this peptide in extracts of guinea pig tissues. In addition to immunoreactive components coeluting with the 3 mammalian tachykinins, substance P (SP), substance K (SK) and NMK, analyses using reverse-phase HPLC revealed immunoreactive peaks coeluting with the C-terminal octapeptide of SK (SK-(3-10)), an N-terminally extended form of SK (),-preprotachykinin-(72-92)amide), and a yet unidentified peak eluting before NMK in the extracts of guinea pig brain and spinal cord. In contrast to the other tachykinins, SP and SK, which were present in high concentrations in extracts of all peripheral and central tissues examined, NMK-like immunoreactivity was detected only in extracts of central tissues. NMK-like immunoreactivity was not detected in extracts of terminal ileum and urinary bladder.

Correspondence: H.-P. Too, Department of Biological Chemistry and Molecular Pharmacology,240 LongwoodAvenue, Boston, MA 02115, U.S.A.

0167-0115/89/$03.50 © 1989 Elsevier Science Publishers B.V. (BiomedicalDivision)

94 Introduction

The tachykinin family ofbioactive peptides shares a common conserved carboxyl-terminal sequence -Phe-X-Gly-Leu-Met-NH2, where X is an aromatic or a branched aliphatic amino acid [ 1,2]. Until recently, substance P (SP), where X is phenylalanine, was thought to be the only mammalian tachykinin [1]. Two other mammalian tachykinins, substance K (SK, or neurokinin A/neuromedin L/neurokinin ~) and neuromedin K (NMK, or neurokinin B/neurokinin ~, both with valine in position X, have since been isolated and identified as decapeptide amides [3-5]. SP and SK are encoded by a single gene which is distinct from that encoding NMK [6-11]. SP and SK have been detected in a variety of central and peripheral tissues [ 12-16]. A number of attempts to raise specific antisera to NMK were unsuccessful [ 13-15]; these antisera showed substantial cross-reaction with SK and cannot be used to measure NMK-like immunoreactivity (NMK-LI) except in conjunction with HPLC. In the present study, the characteristics of a novel and highly specific antiserum to NMK and the use of this assay for measuring NMK-LI in two central (brain and spinal cord) and two peripheral (bladder and ileum) guinea pig tissue extracts are described. In conjunction with reverse-phase high performance liquid chromatography, we have also used radioimmunoassays that are specific for aliphatic residues in position X of the conserved sequence and for SP to examine the possible existence of novel mammalian tachykinins in these tissue extracts.

Materials and Methods

Materials Synthetic peptides were purchased from either Bachem or IAF Biochem International. All peptides were analyzed by reverse phase high-performance liquid chromatography (RP-HPLC) and their concentrations and compositions were determined by amino acid analysis. Monoiodinated Bolton-Hunter (BH) conjugates of SP, SK, NMK and arginine°-NMK (R°NMK) were synthesized by acylation of the parent peptides with N-hydroxysuccinimidyl-3-(4-hydroxyphenyl)propionate and subsequent iodination with Na125I (Amersham) using chloramine-T. Monoiodinated Tyr°-NMK (125IY°NMK) was prepared by oxidative iodination with chloramine-T using tyrosine°NMK as the precursor. All tracers were reduced with mercaptoethanol and purified by RP-HPLC to a specific activity of > 2000 Ci/mmol. Acetic acid, acetonitrile and water were obtained from Baker Chemical. Trifluoroacetic acid (TFA) and carboxypeptidase Y were obtained from Pierce Co. Sep-pak Cls cartridges were obtained from Waters. N M K fragments were generated by enzymatic digestion of synthetic NMK with carboxypeptidase Y, purified by RP-HPLC, and then characterized by amino acid analysis. Pertussis vaccine was obtained from the Massachusetts Public Health Biologic Laboratories, y-preprotachykinin-(72-92) amide was a gift from Dr. J.E. Krause.

95 Antisera NMK-specific antisera were raised against R°NMK (0.76mg) conjugated to succinyl-BSA (5.8 rag) using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (19 mg; manuscript in preparation). Albino male New Zealand White rabbits (2-3 kg) and albino male Hartley guinea pigs (400-500 g) were purchased from Elm Hill, MA. A day before the initial immunization, the animals were injected with pertussis vaccine intramuscularly. Conjugate (0.3 mg) was dissolved in distilled water, emulsified with an equal volume of Freund's complete adjuvant and then injected subcutaneously at multiple sites (approximately 50 #l/site) on the back of each rabbit. Booster doses (0.15 mg) emulsified in Freund's incomplete adjuvant were administered 17, 51 and 90 days after the initial immunization. The animals were bled two weeks after each boost. Guinea pigs were immunized and boosted similarly but with half the doses used for the rabbits. Tissue extractions Albino guinea pigs (Hartley) of either sex (300-400 g) were sacrificed by decapitation. Spinal cord (cervical and thoracic regions), whole brain minus cerebellum, distal ileum and urinary bladder were rapidly dissected on ice, snap-frozen in liquid nitrogen and stored at - 2 0 ° C until use. Extractions using hot acetic acid (1.6M) and the precautions undertaken to ensure minimal oxidation and loss of peptides were carded out as described previously [16]. Tachykinin-like immunoreactivities in the tissue extracts were concentrated on Sep-pak Cls cartridges, eluted and assayed directly or were characterized by RP-HPLC on a Waters ~tBondapak C18 column as described [16]. Radioimmunoassays (RIAs) All RIAs were carried out in sodium phosphate buffer (50 mM, pH 7.5) containing 0.1~o BSA (Sigma, RIA grade). The volume of sarnples was not more than 20~ of the total assay volume (400/A). Plasma-coated charcoal was employed to separate bound from unbound tracer. Immunoreactivity was quantitated from duplicate standard curves included in each assay using a spline function. Details of the RIAs using antisera R91P-2 (for measuring SP-LI) and R93K-6 (for measuring SK-LI) were given in our previous publication [ 16]. In the case of the broad specificity assay G17K-4 (antiserum dilution of 1 : 400000), the intra-assay coefficient of variation of control samples was 3.7~o and the interassay coefficient of variation was 3.6~/o (n = 9). The detection limit at 2 S.D. was 6.4 fmol/tube and the assay showed full cross-reactivity with SK, SK-(2-10), SK-(3-10), SK-(4-10), eledoisin, NMK, 50~/o cross-reactivity with neuropeptide K (NPK) and 7 ~ cross-reactivity with SP. None of the RIAs used in this study cross-reacted (<0.01 ~ ) with unrelated peptides, e.g. bombesin, vasopressin, oxytocin, luteinizing hormone-releasing hormone, thyrotropin-releasing hormone, gastrin-releasing peptide, vasoactive intestinal peptide, angiotensin II, calcitonin generelated peptide, bradykinin, tuftsin, and neurotensin.

96

Results

Two of 3 rabbits and two of 3 guinea pigs immunized produced antisera which bound NMK tracers in preference to BHSP and BHSK. One of the rabbit antisera, R102N-5B, bound 125IY°NMK, B H N M K and BHR°NMK but not BHSP or BHSK (Fig. 1). Displacement of tracer (125IY°NMK) by N-terminal fragments of NMK is shown in Fig. 2. The rank order of potency in displacing the tracer was: NMK > NMK( 1 - 9 ) > N M K - ( 1 - 7 ) > N M K - ( 1 - 6 ) > N M K - ( 1 - 5 ) . This assay (R102N-5B) also showed very low cross-reactivity ( < 0.01 ~o) with either SP or SK (Fig. 3). The interassay and intra-assay coefficients of variation of control samples in this assay (antiserum dilution of 1:40000) were 2.2~o and 5.6~o (n -- 9), respectively. The detection limit at 2 S.D. was 8.6 fmol/tube and the IC5o (concentration of NMK required to inhibit 50~o of the bound tracer) was 25 + 1.4 fmol/tube (n = 9) We have previously examined the utility of neutral water extraction of rat tissues and found it to be rather inefficient in extracting SP-LI, SK-LI and NMK-LI as compared to extraction with aqueous acetic acid [ 16]. We have thus adopted acetic acid extraction as a method of choice in this study. The concentration of SPLI in all 4 tissues examined was found to be substantially higher than SK-LI (Table I). The ratios of SP-LI/SK-LI in the extracts of guinea pig brain, spinal cord, bladder and terminal ileum were found to be about two to 3 (Table II). However, the ratios of SP-LI/NMK-LI in these tissues varied considerably (Table II). The ratios of SP-LI/NMK-LI in the urinary bladder and ileum extracts were found to be greater than 1000 (Table II). The immunoreactivities in the extracts of all 4 tissues were then analyzed by RPHPLC. The ratios of SP-LI/SK-LI and SP-LI/NMK-LI and the concentrations of the

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97

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tachykinin-like immunoreactivities determined from HPLC were similar to those determined by direct measurements of Sep-pak C18 eluates, validating the latter method of measurement. In all the 4 tissue extracts examined, a single immunoreactive peak eluting at the position of synthetic SP was detected by R91P-2 (Figs. 4-7). Similarly, a single immunoreactive peak coeluting with NMK (and minor peaks coeluting with its sulfoxides) was detected by R102N-5B (Figs. 4, 5). Under the extraction conditions employed, oxidation of the immunoreactive peptides was minimal (less than 5 % of total immunoreactivity), thus enabling unambiguous identification of the immunoreactive peaks. Both the G17K-4 and R93K-6 assays showed multiple immunoreactive peaks in all the tissue extracts examined (Figs. 4-7). The peak eluting in fractions 26-28 was clearly

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98

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resolved from synthetic S K or S K sulfoxide. The immunoreactive peak eluting between S K and SP (fractions 3 5 - 3 7 ; Figs. 4 - 7 ) coeluted with synthetic S K - ( 3 - 1 0 ) . A n immunoreactive peak eluting between a m o n o s u l f o x i d e o f N M K and N M K (fractions 5 5 - 5 7 ) was detected by G 1 7 K - 4 and R 9 3 K - 6 assays but not by either R91P-2 (specific assay for SP) or R 1 0 2 N - 5 B (specific assay for N M K ) . Furthermore, this immunoreactive peak did not coelute with porcine neuropeptide K (fractions 6 1 - 6 4 ) or its sulfoxide

99 TABLE I Concentrations of immunoreactive tachykinins in extracts of guinea pig tissues Concentrations are expressed in units of pmol/g wet weight of tissue as means + S.D. of 5 samples in all tissues except whole brain (n = 4). Samples were eluted from the Sep-pak cartridges and assayed directly. Tissues

SP-LI

Whole brain Spinal cord Bladder Ileum

19.9 234.6 20.5 63.9

+ 7.6 + 43.2 _ 0.9 _. 14.5

SK-LI

NMK-LI

7.3 76.3 8.6 20.8

7.2 __ 2.3 14,9 _+ 2.0 * **

-4-_1.7 _+ 8.4 _+ 2.6 _+ 5.8

* No NMK-LI was detected with as much as 50 mg of tissue ( < 0.04 pmol/g wet weight tissue). ** Measured with about 400 mg of tissue (0.04 +_ 0.01 pmol/g wet weight tissue).

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100 TABLE II Ratios of immunoreactive taehykinins in extracts of guinea pig tissues Ratios are expressed as means + S.E.M. of the number of tissues used (in parentheses). Samples were eluted from the Sep-pak cartridges and assayed directly. NMK-LI in guinea pig bladder and ileum was either absent or present in extremely low concentrations (Table I). Tissues

SP-LI/SK-LI

SP-LI/NMK-LI

Whole brain Spinal cord Bladder Ileum

2.54 3.06 2.48 3.12

2.37 + 0.27 (4) 16.20 + 2.14 (5) > 1000 (5) > 1000 (5)

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(fractions 59-62). N o immunoreactive peak eluting in the position of eledoisin was detected by any of the assays used. Guinea pig ileum and urinary bladder extracts contained no N M K - L I (or extremely low concentrations) as measured using R102N-5B (Table I; Figs. 6d, 7d). Radioimmunoassays conducted on very concentrated (1 g tissue equivalent/ml assay buffer) extracts of ileum gave measurable values of N M K - L I (Table I). The extremely low levels of N M K - L I thus detected may be an artifact of non-specific interference at very high protein concentrations. This explanation is consistent with the absence of immunoreactivity coeluting with synthetic N M K in H P L C analyses of these extracts (Figs. 6, 7).

102

Discussion

In this study we have described the development and use of a novel and highly specific RIA for measuring NMK-LI in tissues. NMK-LI appeared to be absent from acetic acid extracts of ileum and bladder. In addition, the presence of tachykinin-immunoreactive peaks coduting with the C-terminal octapoptide of SK (SK-(3-10)), an N-terminally extended form of SK (),-preprotachykinin-(72-92) amide), and a yet to be identified peak eluting before NMK is demonstrated using reverse phase HPLC. Previous attempts by a number of groups to raise specific antisera to NMK have been unsuccessful [ 13-15]. The latter polyclonal antisera showed substantial cross-reaction with SK, which shares with NMK the common C-terminal sequence, -Phe-Val-GlyLeu-Met-NH 2. However, polyclonal antisera to the N-terminal region of NMK have recently been generated using immunogens presenting the N-terminal sequences as the haptenic functional groups [17,18] or by inducing immunotolerance to the common C-terminal epitopes [ 19]. Our polyclonal antiserum R102N-5B appeared to require neither an intact N-terminus nor an intact C-terminus of the peptide for recognition (Figs. 1, 2). Furthermore, the antiserum did not bind BHSK which shares the common C-terminal pentapeptide amide sequence noted above with 125IY°NMK (Fig. 1). In extracts of guinea pig tissues, no immunoreactive components other than those coeluting with synthetic NMK and its sulfoxides were detected with R102N-5B assay (Figs. 4, 5). With an assay using an earlier bleed from the same animal (R102N-3), we have previously shown [ 16] the existence of immunoreactive peaks that coeluted with synthetic NMK and its sulfoxides in rat spinal cord extracts. There is, however, a difference between the ratios of SPLI/NMK-LI in extracts of rat spinal cord (a ratio of 5 [16]) and that in extracts of guinea pig spinal cord (a ratio of 16; Table I). In the guinea pig ileum, all the 3 mammalian tachykinins (SP, SK and NMK) are pharmacologically active [20-23]. NMK has been shown to release acetylcholine causing an atropine- [21,22] and enkephalin- [23]-sensitive contraction in this tissue. This effect was thought to be mediated through a specific 'neuronal substance P receptor' and NMK was proposed as the natural ligand. This hypothesis was further supported by f'mdings using an agonist (senktide) thought to be specific for this subtype of tachykinin receptor [24]. Using the specific NMK assay which we have developed, there is apparently no NMK-LI (or a very low concentration) in the extracts of ileum where SP-LI and SK-LI were readily measured (Figs. 6, 7, Table I). In contrast, NMK-LI as well as SP-LI and SK-LI were readily measured in extracts of brain and spinal cord from the same animals. Furthermore, both B H N M K and 125y°NMK were quantitatively recovered from spiked samples of all 4 tissue extracts after elution from Sep-pak C18 cartridges. The failure to detect NMK-LI in the extracts from ileum is thus not simply due to non-specific loss through hydrophobic adsorption. The apparent absence of NMK in ileum has also been observed previously using highly cross-reactive RIAs in conjunction with gel-permeation chromatography and RP-HPLC [ 13]. These studies and further unpublished work from our laboratory are consistent with the hypothesis that NMK, in contrast to almost all known neuropeptides, is absent from the gut.

103

Recently, a cDNA encoding the precursor of NMK has been isolated and characterized by molecular cloning and sequence analysis from a bovine intestinal cDNA library [8]. RNA blot analysis showed a considerable amount of the preprotachykinin B mRNA (encoding the N M K nucleotide sequences) in the bovine hypothalamus and intestine and none in the cerebral cortex or striatum. This finding is in contrast to a study with rat tissues, where cerebral cortex and striatum contained high amounts of NMK mRNA [6]. The reason for the discrepancy remains to be resolved and the assumption that gastrointestinal tract in rodents is likely to contain N M K mRNA as reported in bovine intestine [6] may not be valid. If the guinea pig intestine does contain NMK mRNA but not NMK, expression of the peptide may be post-translationally controlled. A number of tachykinins have been shown to contract the guinea pig urinary bladder [25]. In vitro binding studies have provided evidence consistent with the proposal that this tissue possesses more than one subtype of tachykinin receptor [26,27]. We have not been able to detect NMK-LI in extracts of this tissue either by direct measurements (Table I) or after RP-HPLC (Fig. 7a, d). Thus, N M K may not be present in peripheral tissues, e.g., the guinea pig urinary bladder and ileum. In the rat nervous system, SP has been shown to be encoded by 3 distinct mRNAs (~, fl and )') derived from the preprotachykinin A gene (PPT-A). The fl- and )'-forms of this mRNA, which each encode a single copy of both SP and SK, have been shown to be the most abundant forms in rat tissues expressing this gene [9]. The ),-form, which accounts for about 80~ of the total PPT mRNA, encodes both SP and SK but not the 36-mer peptide NPK; the fl-form, which encodes SP, SK and NPK accounts for only 20~o of PPT mRNA [28]. Based on these observations, equimolar amounts of SP and SK would be expected to result when either the fl- or )'-form of the PPT mRNA is translated. However, the content of SP is consistently found to be greater than SK in tissue extracts from either guinea pig (Table I [ 13 ]) or rat [ 14,16]. Possible explanations include differential extraction and differential posttranslational processing of these peptides. Multiple tachykinin immunoreactive peaks were detected with either G17K-4 or R93K-6 assays (Figs. 4-7). Preliminary studies showed that the immunoreactivity eluting before synthetic SK (fractions 26-28) coeluted with an N-terminally extended form of SK (~-PPT-(72-92) amide [29]) derived from the ~ form of PPT-A mRNA [7,9]. No immunoreactivity eluting at the position of synthetic porcine NPK [30] was detected by any of the assays used. However, an immunoreactive peak (fraction 55-57) was detected by G17K-4 and R93K-6 assays which does not coelute with any of the known mammalian tachykinins or their sulfoxides (Figs. 4-7). This immunoreactive peak was seen in HPLC chromatograms of tissue extracts from guinea pig (Figs. 4-7) but not from either rat [ 16] or rabbit (unpublished observation). An immunoreactive peak eluting close to but not identical to synthetic porcine NPK has been observed in extracts of guinea pig small intestine [ 13]. Furthermore, under isocratic elution conditions, this immunoreactive peak did not coelute with synthetic porcine NPK. Whether the immunoreactive peak (fraction 55-57) detected in our study is a different form of NPK or another precursor of SK is yet to be determined. No immunoreactivity eluting at the position of eledoisin was detected by any of the RIAs used. Under our HPLC conditions, synthetic eledoisin eluted at a position later

104

than that of synthetic SP (Figs. 4-7). Tachykinin-like immunoreactivity in extracts of mammalian tissues coeluting with eledoisin on RP-HPLC has recently been reported [31-33 ]. In those studies, the purported eledoisin-like immunoreactivity eluted between SK and SP. In the present study, an immunoreactive component eluting between SK and SP was also detected (Figs. 4-7). However, this immunoreactivity coeluted with synthetic SK-(3-10) [16], a truncated form of SK [33]. In conclusion, we have demonstrated the use of a novel and highly specific radioimmunoassay for measuring NMK content in tissue extracts. With this assay, quantitation of NMK in tissue extracts and releases will be possible by direct measurements without the use of highly cross-reactive RIAs in conjunction with chromatography. NMK appears to be absent in extracts of guinea pig ileum and bladder. Guinea pig tissue extracts showed heterogeneity of tachykinin-LI as assayed by the RIAs used in the present study.

Acknowledgements Supported by the National Institutes of Health, Grant NS-22961.

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