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Dynorphin immunoreactivity in the toad neurointermediate lobe
Life Sciences, Vol. 31, pp. 1801-1804 Printed in the U.S.A.
Pergamon Pres~
DYNORPHIN IMMUNOREACTIVITY IN THE TOAD NEUROINTERMEDIATE LOBE Ric I. Cone Addiction Research Foundation,
701 Welch Road, Palo Alto, CA 94304
(Received in final form June 14, 1982) Summary The predominant immunoreactive dynorphin in the toad neurointermediate lobe was similar to that isolated from toad brain with respect to its apparent molecular weight and HPLC retention time. Specific immunoreactive dynorphin staining was present within pars nervosa but not within pars intermedia or pars distalis based on immunohistochemical studies. These results suggest that there is immunoreactive dynorphin in the toad pars nervosa with properties similar to those in toad brain. A biologically-active opioid from toad (Bufo marinus) brain which is immunoreactive with an antiserum specific for dynorphin was recently isolated (I). This opioid, referred to as toad dynorphin, has an apparent molecular weight of 1.4 - 1.8 kDal and resembles porcine dynorphin in having a similar naloxone K e in the guinea pig ileum preparation. Toad dynorphin probably shares considerable sequence homology with porcine dynorphin in addition to having an approximated molecular weight in the same range. However, it differs in having a longer retention time on HPLC and a somewhat lower opioid activity relative to its immunoreactivity. There are very high concentrations of immunoreactive dynorphin (ir-Dyn) in the neurointermediate lobe (NIL) of the toad pituitary, and most of this has an apparent molecular weight of 1.4 to 1.8 kDal (i). This 1.4 - 1.8 kDal ir-Dyn from NIL was characterized on HPLC and found to consist of two peaks; a major peak with a retention time that was the same as that of toad brain dynorphin and a minor peak with a somewhat longer retention time (Figure i). The HPLC purified ir-Dyn from NIL was not fully characterized with regard to its opioid biologic activity because of losses during the purification. However, preliminary studies using the guinea pig ileum preparation indicate that both ir-Dyn peaks contain opioid activity which can be reversed by naloxone. The distribution of ir-Dyn in toad neural tissues is currently under study using immunohistochemical technique. Antisera specific for dynorphin or various fragments of dynorphin showed intense staining of the toad pars nervosa with no staining in either pars intermedia or pars distalis (Figure 2). This immunostaining could be prevented by coincubation of the antisera with dynorphin but not with leucine enkephalin. There are some interesting comparative differences which have emerged from studying dynorphin in the toad brain. Results suggest that the sequence of toad dynorphin differs somewhat f r ~ porcine dynorphin. This difference apparently does not result in a substantial alteration of the biologic 0024-3205/82/161801-04503.00/0 Copyright (c) 1982 Pergamon Press Ltd.
1802
Dynorphin in Toad Neurointermediate
Lobe
Vol. 31, No.s 16 & 17, 1982
12 Pituitary
NIL
8
o
4
v 0
I
I
I
I 29
I 31
I 33
.=
12 Brain 8
4
0
I 27
Acetonltrile
concentration
(%)
FIG. 1 Reverse-phase HPLC of toad pituitary NIL (upper record) and brain (lower record). Tissues were extracted in acetic acid. The extracts were placed on Sephadex G-50 columns and the 1.4 - 1.8 kDal peaks were collected and concentrated using an ion exchange resin. The concentrates were purified by HPLC using a Waters ~Bondapak C18 column (3.9 x 300 ~ ) equilibrated by 25% acetonitrile (vol/vo[) containing 12 mM trifluoroacetic acid. A linear gradient to 35% acetonitrile was introduced to the column over 45 min at 1 ml/min, and fractions (0.6 ml) were collected and assayed for ir-Dyn. Additional details of purification and radioimmunoassay have been described by Cone & Goldstein (i). Concentrates of toad NIL (45.5 pmol ir-Dyn) and brain (54.2 pmol ir-Dyn) were run separately. The major ir-Dyn peaks eluted 27.3 and 27.0 min after the start of the gradient (30.4 and 30.3% acetonitrile) respectively. The second peak of ir-Dyn in the NIL concentrate eluted 32.4 min after the start of the gradient (31.5% acetonitrile). Recovery in the major peak was 6 ~ 5 f o r NIL and 38% for brain. Elution positions of the marker, [~ I]-dynorphin were aligned in the figure for purposes of comparison. The position of porcine dynorphin was also adjusted based on the position of the marker which did not vary significantly over numerous separations.
Vol. 31, No.s 16 & 17, 1982
Dynorphin in Toad Neurointermediate
Lobe
1803
properties which distinguish dynorphin from other classes of opioid peptide. For example, estimates of naloxone K e in the guinea pig ileum preparation suggest that toad dynorphin, like its mammalian counterpart (2), is a kappa receptor agonist. This contention is supported by recent evidence that there are high affinity sites in the toad brain for the kappa agonist, ethylketocyclazocine (3).
A
B FIG. 2
Toad pituitary stained with antiserum raised against dynorphin-(l-13). Paraformaldehyde-fixed tissue was cut into 14 ~ sections in the transverse plane and stained using a double antibody immunofluorescence method (6). (A) The antiserum stained pars nervosa (arrow) but not pars intermedia (double arrow) or pars distalis (not shown). (B) Adjacent section incubated with the same antiserum in the presence of 20 ~M dynorphin. Note the loss of staining in pars nervosa. Bars = 50 ~. Coincubation of this antiserum with leucine enkephalin (20 ~M) did not affect staining, and results similar to those described here were obtained using antisera directed against either dynorphin or dynorphin-(9-17).
The work presented here e: ends the study of ir-Dyn in the toad neurointermediate lobe. Using> an antiserum raised against dynorphin-(l-13), the major species of ir-Dyn is NIL had an apparent molecular weight and retention time on HPLC that was similar to that of toad dynorphin, the major ir-Dyn species in brain. Within NIL, ir-Dyn staining was present within pars nervosa but not within pars intermedia, a result consistent with studies in rat neural lobe (4). The absence of ir-Dyn staining in pars distalis was consistent with previously reported results indicating negligible amounts of ir-Dyn in toad anterior pituitary (i).
1804
Dynorphin in Toad Neurointermediate Lobe
Vol. 31, No.s 16 & 17, 1982
Recently, specific immunostaining with an antiserum directed against dynorphin-(9-17) was observed within a dense bundle of fibers which extend from the pars ventralis of the tuber cinereum into the external zone of the median eminence (manuscript in preparation). Specific, but somewhat less dramatic immunostaining was observed in this same region with other dynorphin antisera. Similar fiber patterns have been observed in the external zone of Rana temporaria median eminence using antisera raised against vasotocin (5). Ir-Dyn has been localized within the supraoptic and paraventricular nuclei of the rat (4,6) and evidence suggests that there are cell bodies in these hypothalamic regions which contain both dynorphin and vasopressin (7). Studies are currently underway to map ir-Dyn perikarya in toad brain. In summary, Bufo marinus is an interesting species for the comparative study of opioid peptides such as dynorphin. By purifying and characterizing these opioids in lower vertebrates it may be possible to reconstruct evolutionary changes in their molecular structure, physiologic action and anatomic localization. Acknowled@ements I thank Drs. Avram Goldstein, Jack Barchas and Eckard Weber for use of their facilities and antisera, and Denise Hirai for her skillful assistance. This work was supported by Grant DA-II99 from the National Institute on Drug Abuse. References i. 2. 3. 4. 5. 6. 7.
R.I. CONE and A. GOLDSTEIN, Proc. Natl. Acad. Sci. USA 79 3345-3349 (1982). C. CHAVKIN, I.F. JAMES and A. GOLDSTEIN, Science 215 413-415 (1982). I.F. JAMES, Life Sci. (submitted for publication). S.J. WATSON, H. AKIL, V.E. GHAZAROSSIAN and A. GOLDSTEIN, Proc. Natl. Acad. Sci. USA 78 1260-1263 (1981). K. DIERICKX and F. VANDESANDE, Cell Tiss. Res. 177 47-56 (1977). E. WEBER, K.A. ROTH and J.D. BARCHAS, Proc. Natl. Acad. Sci. USA 79 30623066 (1982). S.J. WATSON, H. AKIL, W. FISCHLI, A. GOLDSTEIN, E. ZIMMERMAN, G. NILAVER and T.B. VAN WIMERSMA GREIDANUS, Science 216 85-87 (1982).
Pergamon Pres~
DYNORPHIN IMMUNOREACTIVITY IN THE TOAD NEUROINTERMEDIATE LOBE Ric I. Cone Addiction Research Foundation,
701 Welch Road, Palo Alto, CA 94304
(Received in final form June 14, 1982) Summary The predominant immunoreactive dynorphin in the toad neurointermediate lobe was similar to that isolated from toad brain with respect to its apparent molecular weight and HPLC retention time. Specific immunoreactive dynorphin staining was present within pars nervosa but not within pars intermedia or pars distalis based on immunohistochemical studies. These results suggest that there is immunoreactive dynorphin in the toad pars nervosa with properties similar to those in toad brain. A biologically-active opioid from toad (Bufo marinus) brain which is immunoreactive with an antiserum specific for dynorphin was recently isolated (I). This opioid, referred to as toad dynorphin, has an apparent molecular weight of 1.4 - 1.8 kDal and resembles porcine dynorphin in having a similar naloxone K e in the guinea pig ileum preparation. Toad dynorphin probably shares considerable sequence homology with porcine dynorphin in addition to having an approximated molecular weight in the same range. However, it differs in having a longer retention time on HPLC and a somewhat lower opioid activity relative to its immunoreactivity. There are very high concentrations of immunoreactive dynorphin (ir-Dyn) in the neurointermediate lobe (NIL) of the toad pituitary, and most of this has an apparent molecular weight of 1.4 to 1.8 kDal (i). This 1.4 - 1.8 kDal ir-Dyn from NIL was characterized on HPLC and found to consist of two peaks; a major peak with a retention time that was the same as that of toad brain dynorphin and a minor peak with a somewhat longer retention time (Figure i). The HPLC purified ir-Dyn from NIL was not fully characterized with regard to its opioid biologic activity because of losses during the purification. However, preliminary studies using the guinea pig ileum preparation indicate that both ir-Dyn peaks contain opioid activity which can be reversed by naloxone. The distribution of ir-Dyn in toad neural tissues is currently under study using immunohistochemical technique. Antisera specific for dynorphin or various fragments of dynorphin showed intense staining of the toad pars nervosa with no staining in either pars intermedia or pars distalis (Figure 2). This immunostaining could be prevented by coincubation of the antisera with dynorphin but not with leucine enkephalin. There are some interesting comparative differences which have emerged from studying dynorphin in the toad brain. Results suggest that the sequence of toad dynorphin differs somewhat f r ~ porcine dynorphin. This difference apparently does not result in a substantial alteration of the biologic 0024-3205/82/161801-04503.00/0 Copyright (c) 1982 Pergamon Press Ltd.
1802
Dynorphin in Toad Neurointermediate
Lobe
Vol. 31, No.s 16 & 17, 1982
12 Pituitary
NIL
8
o
4
v 0
I
I
I
I 29
I 31
I 33
.=
12 Brain 8
4
0
I 27
Acetonltrile
concentration
(%)
FIG. 1 Reverse-phase HPLC of toad pituitary NIL (upper record) and brain (lower record). Tissues were extracted in acetic acid. The extracts were placed on Sephadex G-50 columns and the 1.4 - 1.8 kDal peaks were collected and concentrated using an ion exchange resin. The concentrates were purified by HPLC using a Waters ~Bondapak C18 column (3.9 x 300 ~ ) equilibrated by 25% acetonitrile (vol/vo[) containing 12 mM trifluoroacetic acid. A linear gradient to 35% acetonitrile was introduced to the column over 45 min at 1 ml/min, and fractions (0.6 ml) were collected and assayed for ir-Dyn. Additional details of purification and radioimmunoassay have been described by Cone & Goldstein (i). Concentrates of toad NIL (45.5 pmol ir-Dyn) and brain (54.2 pmol ir-Dyn) were run separately. The major ir-Dyn peaks eluted 27.3 and 27.0 min after the start of the gradient (30.4 and 30.3% acetonitrile) respectively. The second peak of ir-Dyn in the NIL concentrate eluted 32.4 min after the start of the gradient (31.5% acetonitrile). Recovery in the major peak was 6 ~ 5 f o r NIL and 38% for brain. Elution positions of the marker, [~ I]-dynorphin were aligned in the figure for purposes of comparison. The position of porcine dynorphin was also adjusted based on the position of the marker which did not vary significantly over numerous separations.
Vol. 31, No.s 16 & 17, 1982
Dynorphin in Toad Neurointermediate
Lobe
1803
properties which distinguish dynorphin from other classes of opioid peptide. For example, estimates of naloxone K e in the guinea pig ileum preparation suggest that toad dynorphin, like its mammalian counterpart (2), is a kappa receptor agonist. This contention is supported by recent evidence that there are high affinity sites in the toad brain for the kappa agonist, ethylketocyclazocine (3).
A
B FIG. 2
Toad pituitary stained with antiserum raised against dynorphin-(l-13). Paraformaldehyde-fixed tissue was cut into 14 ~ sections in the transverse plane and stained using a double antibody immunofluorescence method (6). (A) The antiserum stained pars nervosa (arrow) but not pars intermedia (double arrow) or pars distalis (not shown). (B) Adjacent section incubated with the same antiserum in the presence of 20 ~M dynorphin. Note the loss of staining in pars nervosa. Bars = 50 ~. Coincubation of this antiserum with leucine enkephalin (20 ~M) did not affect staining, and results similar to those described here were obtained using antisera directed against either dynorphin or dynorphin-(9-17).
The work presented here e: ends the study of ir-Dyn in the toad neurointermediate lobe. Using> an antiserum raised against dynorphin-(l-13), the major species of ir-Dyn is NIL had an apparent molecular weight and retention time on HPLC that was similar to that of toad dynorphin, the major ir-Dyn species in brain. Within NIL, ir-Dyn staining was present within pars nervosa but not within pars intermedia, a result consistent with studies in rat neural lobe (4). The absence of ir-Dyn staining in pars distalis was consistent with previously reported results indicating negligible amounts of ir-Dyn in toad anterior pituitary (i).
1804
Dynorphin in Toad Neurointermediate Lobe
Vol. 31, No.s 16 & 17, 1982
Recently, specific immunostaining with an antiserum directed against dynorphin-(9-17) was observed within a dense bundle of fibers which extend from the pars ventralis of the tuber cinereum into the external zone of the median eminence (manuscript in preparation). Specific, but somewhat less dramatic immunostaining was observed in this same region with other dynorphin antisera. Similar fiber patterns have been observed in the external zone of Rana temporaria median eminence using antisera raised against vasotocin (5). Ir-Dyn has been localized within the supraoptic and paraventricular nuclei of the rat (4,6) and evidence suggests that there are cell bodies in these hypothalamic regions which contain both dynorphin and vasopressin (7). Studies are currently underway to map ir-Dyn perikarya in toad brain. In summary, Bufo marinus is an interesting species for the comparative study of opioid peptides such as dynorphin. By purifying and characterizing these opioids in lower vertebrates it may be possible to reconstruct evolutionary changes in their molecular structure, physiologic action and anatomic localization. Acknowled@ements I thank Drs. Avram Goldstein, Jack Barchas and Eckard Weber for use of their facilities and antisera, and Denise Hirai for her skillful assistance. This work was supported by Grant DA-II99 from the National Institute on Drug Abuse. References i. 2. 3. 4. 5. 6. 7.
R.I. CONE and A. GOLDSTEIN, Proc. Natl. Acad. Sci. USA 79 3345-3349 (1982). C. CHAVKIN, I.F. JAMES and A. GOLDSTEIN, Science 215 413-415 (1982). I.F. JAMES, Life Sci. (submitted for publication). S.J. WATSON, H. AKIL, V.E. GHAZAROSSIAN and A. GOLDSTEIN, Proc. Natl. Acad. Sci. USA 78 1260-1263 (1981). K. DIERICKX and F. VANDESANDE, Cell Tiss. Res. 177 47-56 (1977). E. WEBER, K.A. ROTH and J.D. BARCHAS, Proc. Natl. Acad. Sci. USA 79 30623066 (1982). S.J. WATSON, H. AKIL, W. FISCHLI, A. GOLDSTEIN, E. ZIMMERMAN, G. NILAVER and T.B. VAN WIMERSMA GREIDANUS, Science 216 85-87 (1982).