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Distribution of Met5-enkephalin-Arg6,Phe7 in rat spinal cord
Brain Rc~earck, 264 (i9~3) 330 ~39
336
I lsevier Bi¢~medicai Pre~
Distribution of Mets-enkephalin-Arg6,Phe ' in rat spinal cord E, A. MAJAN E, M. J. I A D A R O L A and H.-Y. T. YANG*
Laboratory of Prectinical Pharmacologr, National Institute of Mental Health, Saint Elizabeths Hospital, Washington, D.C. 20032 (U.S.A.) (Accepted November 23rd, 1982)
Key words: spinal cord - MetS-enkephalin - Met 5-enkephalin-ArgS,Phe 7
opioid peptide - neuropeptides
The distribution of MetS-enkephalin-Arf,Phd ( Y G G F M R F ) in rat spinal cord was determined by a specific RIA and compared with that of MetS-enkephalin. The concentration of Y G G F M R F on a per mg protein basis was highest in sacral cord and successively decreased in more rostral segments. A similar rostro,caudal distribution was observed for Met 5-enkephalin. Regional microdissection revealed the dorsal grey matter to be highest in Y G G F M R F content followed by ventral gray, ventral white and dorsal white matter: a similar pattern was observed for Met-~-enkephalin. The ratio of MetS-enkephalin: Y G G F M R F concentration was 5.4 +_ 0.15 (S.E.M.) on average in all regions measured, indicating a very close quantitative relationship between the two molecules. Our data suggest that Y G G F M R F may act as a precursor of or cotransmitter with MetS-enkephalin in spinal cord tissue.
Me¢-enkephalin-Arg6,Phd ( Y G G F M R F ) was first isolated from bovine adrenal glands in 1979 I:. Recently, Y G G F M R F was found to be unevenly distributed in various brain structures but this pattern was parallel with the distribution of MetS-enkephalin ~~7. Furthermore. in every brain region analyzed, the concentration of Y G G F M R F was found to be lower than that of Me¢-enkephalin ~,~7. This pattern of distribution is compatible with the view that Y G G F M R F may function either as a precursor of MetS-en kephalin or as a cotransmitter of Met 5-enkephalin. The latter possibility is supported by the observation that Y G G F M R F has opiate-like activity in various biological systems6,9 and can be released from brain slices j7 by K + depolarization in a Ca:+-dependent manner. The peptide Y G G F M R F , is also widely distributed in peripheral tissues and, in some of them such as the lung, the content of Y G G F M R F is much greater than that of MetS-enkephalin ~3. Recently, the existence of Y G G F M R F in the spinal cord was detected immunohistochemically ~4. Since a detailed quantitative distribution of Y G G F M R F * To whom correspondence should be addressed.
0006-8993/83/000(~0000/$03,00 ~,;1983 Elsevier Science Publishers
within the spinal cord was not available, we have studied the rostro-caudal and dorsal-yen trat distribution of Y G G F M R F in rat spinal cord by radioimmunoassay and compared this pattern with the distribution of MetS-enkephalin. Male Sprague-Dawley rats (400 500 g) were decapitated and the spinal cords removed rapi d l f . For the rostro-caudal distribution of Y G G F M R F , the spinal cords were divided, while on ice, into cervical, thoracic, lumbar and sacral regions. For the dorso-ventral distribution, the spinal cords were sliced with a razor blade and dissected on a cold plate (0 °C) into dorsal gray, dorsal white, ventral gray and ventral white areas. The samples were immediately crushed in an ice-cold solution of 1 N CH3COOH, 0.02 N HCI and 0.1%/~-mercaptoethanol in polypropylene tubes. After the completion of the dissection, the tissues were homogenized by sonication and the homogenates centrifuged at 18,000 g for 20 min. The supernatants were lyophilized, redissolved in H20 and analyzed by radioimmunoassay for MetS-enkephalin ~t' and Y G G F M R F ~7as previously described.
337 TABLE 1
Rostro-caudal distribution of YGGFMRF and Met -~-enkephalin in rat spinal cord Results indicate the total immunoreactive material and are expressed as p m o l / m g protein, m e a n ± S.E.M. (n = 9 10). Protein was determined according to t . o w ~ et al. 6. with bovine s e r u m a l b u m i n as standard.
Spinal sef, ment
Y GG FM RF (pmol/mgprotein)
Met 5 -enkephalin Ipmol/mgprotein)
Mers-enkephalin YGGFMRF
Cervical Thoracic Lumbar Sacral
0.92 0.94 1.4 2.3
5.3 4.9 8.3 13.0
5.8 5.2 5.9 5.7
_+ 0.08 + 0.09 + 0.1 _+ 0.08
_+ 0.5 _ 0.7 ± 0.8 ± 0.9
The content of Y G G F M R F and Met-~-enkephalin immunoreactivities is higher in the caudal than in the rostral regions of the spinal cord; the content of Y G G F M R F immunoreactivity in the sacral region is about twice that in the cervical or thoracic region (Table I). An even more striking specific distribution is observed dorso-ventrally. The concentrations are higher in gray than in white matter and the highest value observed is in the dorsal gray matter where the Y G G F M R F immunoreactivity is about 6 7 times more concentrated than in white matter (Table II). The distribution of MetS-enkephalin follows closely that of Y G G F M R F (Tables I and lI). The level of Y G G F M R F immunoreactivity is lower than that of Met~-enkephalin and the ratio of Met Se n k e p h a l i n / Y G G F M R F appears to be constant in every region analyzed. Such a parallel distribution of these two opioid peptides has also been observed in various brain regions ~-~7.This may be a reflection of the presence of a preproenkephalin which from studies in adrenal gland can be inferred to contain one copy of Y G G F M R F and 4 copies of MetS-enkephalin4.~.
Interestingly, such a constant ratio in the content of Y G G F M R F and MetS-enkephalin was not observed in peripheral organs where the ratio of MetS-enkephalin/YGGFMRF seems to vary greatly ~3. The specificity of the antisera used in this study has been previously described L6-~7.Briefly, the Y G G F M R F antiserum shows no cross-reactivity with MetS-enkephalin, MetS-enkephalin-Arg~' or MetS-enkephalin-Arg~Arg7 but cross reacts very slightly with the molluscan cardioexcitatory peptide Phe-Met-Arg-Phe-NH, (about 0 . 0 3 % ) 17. The specificity of the Y G G F M R F antiserum was further examined by chromatographic characterization of Y G G F M R F immunoreactivity. The extract from whole spinal cords was fractionated by BioGel P-2 column chromatography. The fractions were lyophilized, redissolved in H~O and then radioimmunoassayed for Y G G F M R F immunoreactivity. More than 80% of the total Y G G F M R F immunoreactivity coeluted with synthetic Y G G F M R F from the BioGel P-2 column (Fig. l): the remaining immunoreactivity was eluted in 3 other minor peaks.
T A B L E II
Dorso-ventral distribution ()/ YGGFMRF and Me/-enkephalin in rat spinal cord Results indicate the total immunoreactive material and are expressed as p m o t / m g protein, m e a n + S.E.M. (n --- 6). Protein was determined as in Table 1.
Re~ion
YGGFMRF (pmol/mgprotein)
Mefi-enkeDhalin (prnol/mgprotein)
Met ~-enkephalin YGGFMRF
Dorsalwhite Dorsal gray Ventral white Vcntralgray
0.76 5.0 1.0 2.9
3.7 22 6.1 15
4.8 4.4 6.1 5.2
± 0.1 _ 0.5 ___ 0.1 -+ 0.2
± _. _ ±
0.8 3 0.9 l
338 Met-Enk-Arg-Phe
Vo
7.5 "-6 E
5.o g
<, ~' Z 5
.........
10
20 Fraction (ml)
30
40
Fig. 1. BioGel P-2 column chromatography of extract from spinal cord. The tissue extract in 0.5 ml 1 N CH3COOH (from 30 mg protein of rat spinal cord) was applied to BioGel P-2 column, 0.9 X 60 cm. The column was then eluted with 1 N CH3COOH at flow rate of 0.1 ml/min. One ml fractions were collected, lyophilized, redissolved in H:O and aliquots were radioimmunoassayed. Results were expressed as pmol MetS-enkephalin-Arg6,Phe7 per ml eluate.
Met-Enk-Arg-Phe
" 1.5
Phe-Met-Arg-P!
-6 E Mel-Enk
?_ ~ 1.0 <
Met-Enk-Arg
0.5
,I
1o
i
20 Fraction (ml)
30
40
Fig. 2. Identification of MetS-enkephalin-Arg~,Phe7 by high pressure liquid chromatography. Fractions 35-42 from BioGel P-2 column (Fig. 1) were combined, lyophilized and applied to a BioSil ODS-10 column, 250 x 4 mm column. The column was eluted at flow rate of 1 m l / m i n with a linear gradient ofacetonitrile, 20-40% in 0.1% trifluoroacetic acid over a period of 40 rain. One ml fractions were collected every minute, lyophilized, redissolved in H 2 0 and radioimmunoassayed. The results are expressed as p m o l / m l eluate. The sample applied is equivalent to an extract from 30 mg protein of rat spinal cord.
For further identification, the main peak t~l" immunoreactive material (fractions 35 42 from BioGel P-2 column (Fig. I )) was fractionatcd by high pressure liquid chromatography using ~l BioSil ODS-10 column. The major portion ~>t" immunoreactive material again coeluted with authentic Y G G F M R F . The nature of the minor immunoreactive components from both columns was not characterized further in this study. The specificity of the antiserum combined with the results of chromatographic characterization (Figs. 1 and 2) strongly indicate that the Y G G F M R F immunoreactivity measured (Tables 1 and Ill was mainly due to authentic Y G G F M R F . The specificity of Met>-enkepha lin antiserum used in this study has also been previously described ~7. The antiserum is directed against the C-terminus of Met~-enkephalin as it cross reacts only slightly with Leu"-enkephalin (about 10%) or MetS-enkephalin-Arg~ and Y G G F M R F (10%). Since the level of YGG FM RF is much lower than that of MetS-enkephalin, the values of MetS-enkephalin immunoreactivities (Tables 1 and lI) should not be significantly affected by the small cross-reaction between Met~-en kephalin antiserum and Y G G F M R F . The finding that MetS-enkephalin is highly concentrated in the dorsal gray (Table Ill is consistent with the previous results o f i m m u n o h i s t o chemical studies showing that Met~-enkephalin immunoreactivity is concentrated in laminae I and II 3~(~-~. In this region, enkephalin is known to be important for the control of nociceptive transmission 3.5-j-~. Similarly to Met~-enkephalin, Y G G F M R F immunoreactivity is most concentrated in the dorsal gray region, however, whether Met-~-enkephalin and Y G G F M R F are stored in the same or different neurons still is currently being pursued using an immunohistochemical approach. The analgesic potency of Y G G F M R F was found to be 8 times greater than that of MetS-enkephalin ~ and the opiate receptors are concentrated in the dorsa! horn ~, These two observations taken together with the distribution of Y G G F M R F (Table I1) ~trongly suggest that this opioid heptapeptide may play an important role in modulating nociceptive transmission in the spinal cord.
339 1 Boarder, M. R., Lockfeld, A. J. and Barchas, J., Measurement of methionine-enkephalin [Arg,6, PheT], in rat brain by specific radioimmunoassay directed at methionine sulphoxide enkephalin [Arf, PheT], J. Neurochem.. 38 (1982) 29% 304. 2 DeSousa. B. N. and Horrocks. k. A., Development of rat spinal cord, Dev. Neurosci.. 2 (1979) 115- 121. 3 Fields. H. k., Emson, P. C., Leigh, B. K., Gilbert, R. F. T. and Iversen, L. L., Multiple opiate receptor sites on primary afferent fibres, Nature (Lond.). 284 (1980) 351 353. 4 Gubler, U., Seeburg, P., Hoffman, B. J., Gage, k. D. and Udenfriend, S., Molecular cloning establishes proenkephalin as precursor of enkephalin-containing peptides, Nature (Lond.), 295 (1982) 206-208. 5 H6kfelt, T., Ljunghdahl, A., Terenius, k., Elde, R. and Nilsson, G., lmmunohistochemical analysis of peptide pathways possibly related to pain and analgesia: enkephalin and substance P, Proe. nat. Acad, Sci. U.S.A.. 74 (1977) 3081 3085. 6 Inturrisi, C. E., Umans, J. G., Wolff, D., Stern, A., Lewis, R. V.. Stein, S. and Udenfriend, S., Analgesic activity of the naturally occurring heptapeptide [met]enkephalinArg~'-Phe 7, Proe. nat. Acad. Sei. U.S.A., 77 (1980) 5512 5514. 7 Lowly, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 ( 1951) 265 275. 8 Noda, M., Furutani, Y.,Takahashi, H., Toyosato, M., Hirose, T., lnayama. S., Nakanishi, S. and Numa, S., Cloning and sequence analysis ofcDNA for bovine adrenal preproenkephalin, Nature (Lond), 295 (1982) 202 206. 9 Rossier, J., Audiger, Y., Ling, N.. Cros, J. and Uden-
10
11
12
13 14 15 16 17
friend, S., Met-enkephalin-Arg6-Phe7. present in high amounts in brain of rat, cattle and man, is an opioid agonist, Nature (Lond.), 288 (1980) 8 8 90. Sar, M., Stumpf, W., Miller, R.,Chang, K.-J. and Cuatrecasas, P., Immunohistochemical localization of enkephalin in rat brain and spinal cord, J. comp. Neurol., 182 (1978) 17 38. Simantov, R., Kuhar, M., Uhl, G. and Snyder, S. H., Opioid peptide enkephalin:immunohistochemical mapping in rat central nervous system, Proc. nat. A cad. Sei. U.S.A., 74(1977)2167 2171. Stern, A. S., Lewis, R. V., Kimura, S., Rossier, J., Gerber, L. D., Brink, L., Stein, S. and Udenfriend, S., Isolation of the opioid heptapeptide Met-enkephalin [Arf,Phe 7] from bovine adrenal medullary granules and striatum, Proc. nat. Acad. Sci. U.S.A.. 76 (1979) 668(~6683. Tang, J., Yang, H.-Y. T. and Costa, E., Distribution of Met-enkephalin-Arff-Phe7 in various tissues of rats and guinea pigs, Neuropharmacologv, 21 (1982) 595 597. Weber, E., Evans, C. J., Samuelsson, S. J. and Barchas, J. D., Novel peptide neuronal system in rat brain and pituitaly, Science, 214(1981) 1248 1251. Woolf, C. J. and Fitzgerald, M., Do opioid peptides mediate a presynaptic control of C-fiber transmission in the rat spinal cord, Neurosci. Lett., 29 (1982) 67 72. Yang, H.-Y. T., Hong, J. S. and Costa, E.. Regional distribution of leu and met-enkephalin in rat brain, Neuropharmacology', 16 (1977) 303 307. Yang, H.-Y. T., Panula,.P., Tang, J. and Costa, E., Characterization and location of[metS]-enkephalin-arg6-phe7 stored in various rat brain regions, J. Neurochem., in press.
336
I lsevier Bi¢~medicai Pre~
Distribution of Mets-enkephalin-Arg6,Phe ' in rat spinal cord E, A. MAJAN E, M. J. I A D A R O L A and H.-Y. T. YANG*
Laboratory of Prectinical Pharmacologr, National Institute of Mental Health, Saint Elizabeths Hospital, Washington, D.C. 20032 (U.S.A.) (Accepted November 23rd, 1982)
Key words: spinal cord - MetS-enkephalin - Met 5-enkephalin-ArgS,Phe 7
opioid peptide - neuropeptides
The distribution of MetS-enkephalin-Arf,Phd ( Y G G F M R F ) in rat spinal cord was determined by a specific RIA and compared with that of MetS-enkephalin. The concentration of Y G G F M R F on a per mg protein basis was highest in sacral cord and successively decreased in more rostral segments. A similar rostro,caudal distribution was observed for Met 5-enkephalin. Regional microdissection revealed the dorsal grey matter to be highest in Y G G F M R F content followed by ventral gray, ventral white and dorsal white matter: a similar pattern was observed for Met-~-enkephalin. The ratio of MetS-enkephalin: Y G G F M R F concentration was 5.4 +_ 0.15 (S.E.M.) on average in all regions measured, indicating a very close quantitative relationship between the two molecules. Our data suggest that Y G G F M R F may act as a precursor of or cotransmitter with MetS-enkephalin in spinal cord tissue.
Me¢-enkephalin-Arg6,Phd ( Y G G F M R F ) was first isolated from bovine adrenal glands in 1979 I:. Recently, Y G G F M R F was found to be unevenly distributed in various brain structures but this pattern was parallel with the distribution of MetS-enkephalin ~~7. Furthermore. in every brain region analyzed, the concentration of Y G G F M R F was found to be lower than that of Me¢-enkephalin ~,~7. This pattern of distribution is compatible with the view that Y G G F M R F may function either as a precursor of MetS-en kephalin or as a cotransmitter of Met 5-enkephalin. The latter possibility is supported by the observation that Y G G F M R F has opiate-like activity in various biological systems6,9 and can be released from brain slices j7 by K + depolarization in a Ca:+-dependent manner. The peptide Y G G F M R F , is also widely distributed in peripheral tissues and, in some of them such as the lung, the content of Y G G F M R F is much greater than that of MetS-enkephalin ~3. Recently, the existence of Y G G F M R F in the spinal cord was detected immunohistochemically ~4. Since a detailed quantitative distribution of Y G G F M R F * To whom correspondence should be addressed.
0006-8993/83/000(~0000/$03,00 ~,;1983 Elsevier Science Publishers
within the spinal cord was not available, we have studied the rostro-caudal and dorsal-yen trat distribution of Y G G F M R F in rat spinal cord by radioimmunoassay and compared this pattern with the distribution of MetS-enkephalin. Male Sprague-Dawley rats (400 500 g) were decapitated and the spinal cords removed rapi d l f . For the rostro-caudal distribution of Y G G F M R F , the spinal cords were divided, while on ice, into cervical, thoracic, lumbar and sacral regions. For the dorso-ventral distribution, the spinal cords were sliced with a razor blade and dissected on a cold plate (0 °C) into dorsal gray, dorsal white, ventral gray and ventral white areas. The samples were immediately crushed in an ice-cold solution of 1 N CH3COOH, 0.02 N HCI and 0.1%/~-mercaptoethanol in polypropylene tubes. After the completion of the dissection, the tissues were homogenized by sonication and the homogenates centrifuged at 18,000 g for 20 min. The supernatants were lyophilized, redissolved in H20 and analyzed by radioimmunoassay for MetS-enkephalin ~t' and Y G G F M R F ~7as previously described.
337 TABLE 1
Rostro-caudal distribution of YGGFMRF and Met -~-enkephalin in rat spinal cord Results indicate the total immunoreactive material and are expressed as p m o l / m g protein, m e a n ± S.E.M. (n = 9 10). Protein was determined according to t . o w ~ et al. 6. with bovine s e r u m a l b u m i n as standard.
Spinal sef, ment
Y GG FM RF (pmol/mgprotein)
Met 5 -enkephalin Ipmol/mgprotein)
Mers-enkephalin YGGFMRF
Cervical Thoracic Lumbar Sacral
0.92 0.94 1.4 2.3
5.3 4.9 8.3 13.0
5.8 5.2 5.9 5.7
_+ 0.08 + 0.09 + 0.1 _+ 0.08
_+ 0.5 _ 0.7 ± 0.8 ± 0.9
The content of Y G G F M R F and Met-~-enkephalin immunoreactivities is higher in the caudal than in the rostral regions of the spinal cord; the content of Y G G F M R F immunoreactivity in the sacral region is about twice that in the cervical or thoracic region (Table I). An even more striking specific distribution is observed dorso-ventrally. The concentrations are higher in gray than in white matter and the highest value observed is in the dorsal gray matter where the Y G G F M R F immunoreactivity is about 6 7 times more concentrated than in white matter (Table II). The distribution of MetS-enkephalin follows closely that of Y G G F M R F (Tables I and lI). The level of Y G G F M R F immunoreactivity is lower than that of Met~-enkephalin and the ratio of Met Se n k e p h a l i n / Y G G F M R F appears to be constant in every region analyzed. Such a parallel distribution of these two opioid peptides has also been observed in various brain regions ~-~7.This may be a reflection of the presence of a preproenkephalin which from studies in adrenal gland can be inferred to contain one copy of Y G G F M R F and 4 copies of MetS-enkephalin4.~.
Interestingly, such a constant ratio in the content of Y G G F M R F and MetS-enkephalin was not observed in peripheral organs where the ratio of MetS-enkephalin/YGGFMRF seems to vary greatly ~3. The specificity of the antisera used in this study has been previously described L6-~7.Briefly, the Y G G F M R F antiserum shows no cross-reactivity with MetS-enkephalin, MetS-enkephalin-Arg~' or MetS-enkephalin-Arg~Arg7 but cross reacts very slightly with the molluscan cardioexcitatory peptide Phe-Met-Arg-Phe-NH, (about 0 . 0 3 % ) 17. The specificity of the Y G G F M R F antiserum was further examined by chromatographic characterization of Y G G F M R F immunoreactivity. The extract from whole spinal cords was fractionated by BioGel P-2 column chromatography. The fractions were lyophilized, redissolved in H~O and then radioimmunoassayed for Y G G F M R F immunoreactivity. More than 80% of the total Y G G F M R F immunoreactivity coeluted with synthetic Y G G F M R F from the BioGel P-2 column (Fig. l): the remaining immunoreactivity was eluted in 3 other minor peaks.
T A B L E II
Dorso-ventral distribution ()/ YGGFMRF and Me/-enkephalin in rat spinal cord Results indicate the total immunoreactive material and are expressed as p m o t / m g protein, m e a n + S.E.M. (n --- 6). Protein was determined as in Table 1.
Re~ion
YGGFMRF (pmol/mgprotein)
Mefi-enkeDhalin (prnol/mgprotein)
Met ~-enkephalin YGGFMRF
Dorsalwhite Dorsal gray Ventral white Vcntralgray
0.76 5.0 1.0 2.9
3.7 22 6.1 15
4.8 4.4 6.1 5.2
± 0.1 _ 0.5 ___ 0.1 -+ 0.2
± _. _ ±
0.8 3 0.9 l
338 Met-Enk-Arg-Phe
Vo
7.5 "-6 E
5.o g
<, ~' Z 5
.........
10
20 Fraction (ml)
30
40
Fig. 1. BioGel P-2 column chromatography of extract from spinal cord. The tissue extract in 0.5 ml 1 N CH3COOH (from 30 mg protein of rat spinal cord) was applied to BioGel P-2 column, 0.9 X 60 cm. The column was then eluted with 1 N CH3COOH at flow rate of 0.1 ml/min. One ml fractions were collected, lyophilized, redissolved in H:O and aliquots were radioimmunoassayed. Results were expressed as pmol MetS-enkephalin-Arg6,Phe7 per ml eluate.
Met-Enk-Arg-Phe
" 1.5
Phe-Met-Arg-P!
-6 E Mel-Enk
?_ ~ 1.0 <
Met-Enk-Arg
0.5
,I
1o
i
20 Fraction (ml)
30
40
Fig. 2. Identification of MetS-enkephalin-Arg~,Phe7 by high pressure liquid chromatography. Fractions 35-42 from BioGel P-2 column (Fig. 1) were combined, lyophilized and applied to a BioSil ODS-10 column, 250 x 4 mm column. The column was eluted at flow rate of 1 m l / m i n with a linear gradient ofacetonitrile, 20-40% in 0.1% trifluoroacetic acid over a period of 40 rain. One ml fractions were collected every minute, lyophilized, redissolved in H 2 0 and radioimmunoassayed. The results are expressed as p m o l / m l eluate. The sample applied is equivalent to an extract from 30 mg protein of rat spinal cord.
For further identification, the main peak t~l" immunoreactive material (fractions 35 42 from BioGel P-2 column (Fig. I )) was fractionatcd by high pressure liquid chromatography using ~l BioSil ODS-10 column. The major portion ~>t" immunoreactive material again coeluted with authentic Y G G F M R F . The nature of the minor immunoreactive components from both columns was not characterized further in this study. The specificity of the antiserum combined with the results of chromatographic characterization (Figs. 1 and 2) strongly indicate that the Y G G F M R F immunoreactivity measured (Tables 1 and Ill was mainly due to authentic Y G G F M R F . The specificity of Met>-enkepha lin antiserum used in this study has also been previously described ~7. The antiserum is directed against the C-terminus of Met~-enkephalin as it cross reacts only slightly with Leu"-enkephalin (about 10%) or MetS-enkephalin-Arg~ and Y G G F M R F (10%). Since the level of YGG FM RF is much lower than that of MetS-enkephalin, the values of MetS-enkephalin immunoreactivities (Tables 1 and lI) should not be significantly affected by the small cross-reaction between Met~-en kephalin antiserum and Y G G F M R F . The finding that MetS-enkephalin is highly concentrated in the dorsal gray (Table Ill is consistent with the previous results o f i m m u n o h i s t o chemical studies showing that Met~-enkephalin immunoreactivity is concentrated in laminae I and II 3~(~-~. In this region, enkephalin is known to be important for the control of nociceptive transmission 3.5-j-~. Similarly to Met~-enkephalin, Y G G F M R F immunoreactivity is most concentrated in the dorsal gray region, however, whether Met-~-enkephalin and Y G G F M R F are stored in the same or different neurons still is currently being pursued using an immunohistochemical approach. The analgesic potency of Y G G F M R F was found to be 8 times greater than that of MetS-enkephalin ~ and the opiate receptors are concentrated in the dorsa! horn ~, These two observations taken together with the distribution of Y G G F M R F (Table I1) ~trongly suggest that this opioid heptapeptide may play an important role in modulating nociceptive transmission in the spinal cord.
339 1 Boarder, M. R., Lockfeld, A. J. and Barchas, J., Measurement of methionine-enkephalin [Arg,6, PheT], in rat brain by specific radioimmunoassay directed at methionine sulphoxide enkephalin [Arf, PheT], J. Neurochem.. 38 (1982) 29% 304. 2 DeSousa. B. N. and Horrocks. k. A., Development of rat spinal cord, Dev. Neurosci.. 2 (1979) 115- 121. 3 Fields. H. k., Emson, P. C., Leigh, B. K., Gilbert, R. F. T. and Iversen, L. L., Multiple opiate receptor sites on primary afferent fibres, Nature (Lond.). 284 (1980) 351 353. 4 Gubler, U., Seeburg, P., Hoffman, B. J., Gage, k. D. and Udenfriend, S., Molecular cloning establishes proenkephalin as precursor of enkephalin-containing peptides, Nature (Lond.), 295 (1982) 206-208. 5 H6kfelt, T., Ljunghdahl, A., Terenius, k., Elde, R. and Nilsson, G., lmmunohistochemical analysis of peptide pathways possibly related to pain and analgesia: enkephalin and substance P, Proe. nat. Acad, Sci. U.S.A.. 74 (1977) 3081 3085. 6 Inturrisi, C. E., Umans, J. G., Wolff, D., Stern, A., Lewis, R. V.. Stein, S. and Udenfriend, S., Analgesic activity of the naturally occurring heptapeptide [met]enkephalinArg~'-Phe 7, Proe. nat. Acad. Sei. U.S.A., 77 (1980) 5512 5514. 7 Lowly, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 ( 1951) 265 275. 8 Noda, M., Furutani, Y.,Takahashi, H., Toyosato, M., Hirose, T., lnayama. S., Nakanishi, S. and Numa, S., Cloning and sequence analysis ofcDNA for bovine adrenal preproenkephalin, Nature (Lond), 295 (1982) 202 206. 9 Rossier, J., Audiger, Y., Ling, N.. Cros, J. and Uden-
10
11
12
13 14 15 16 17
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