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A slow and a fast secretory compartment of POMC-derived peptides in the neurointermediate lobe of the amphibian Xenopus laevis
Camp. Biochem. Physiol. Vol. 96C, No. I, pp. 199-203, 1990 Printed in Great Britain
0306~4492/90 $3.00+ 0.00 0 1990Pergamon Press plc
A SLOW AND A FAST SECRETORY COMPARTMENT OF POMC-DERIVED PEPTIDES IN THE NEUROINTERMEDIATE LOBE OF THE AMPHIBIAN XENOPUS INGRID D. VAN ZOEST, HANS J.
LAEVIS
LEENDERS, BRUCEG. JENKSand ERIC W. ROUBOS
Department of Animal Physiology, Faculty of Science, Catholic University of Nijmegen, Toernooiveld, 6525 ED Nijmegen, The Netherlands. Telephone: (080) 652036, Fax: (080) 553450 (Received 24 January 1990)
Peptide release from the neurointermediate lobe of Xenopus lueuishas been studied using dual pulse-chase incubation, superfusion and HPLC techniques. 2. Lobes release pulse-labelled material in two phases, the first phase lasting about 6 hr, the second persisting up to 14 hr. 3. In both phases similar, POMC-derived peptides are released. Their release can be inhibited by dopamine. 4. When release during the first phase is inhibited, newly synthesized peptides are shunted into the second release pathway. 5. It is concluded that the neurointermediate lobe contains two release compartments. The possible locations of these compartments within melanotrope cells have been discussed. Abstract-l.
INTRODUCTION The biological action of endocrine cells producing the precursor protein proopiomelanocortin (POMC) is determined by the mode of processing of POMC and by the dynamics of the release of POMC-derived peptides. The direction of POMC processing is largely cell-specific. For example, corticotrope cells of the pars distalis of the pituitary gland produce ACTH as a major secretory product, whereas in the melanotrope cells of the pars intermedia, ACTH is further processed to form cr-MSH as a secretory product (Eipper and Mains, 1980; Herbert, 1981). However, within a given cell type, the mode of POMC processing may be, to a certain extent, open to modulation. Much of the evidence for this comes from in vitro experiments, in which POMC cells were treated with pharmacological agents (Loh and Jenks, 1981; Loh et al., 1981; Randle et al., 1983; Goldman and Loh, 1984; Ham and Smyth, 1984; Ham et al., 1984), but in the case of the melanotropes of the South African clawed toad Xenopus laevis it has been shown that a physiological stimulus (background adaptation) can alter POMC processing and, hence, the in vivo profile of the released peptides (Verberg-van Kemenade et al., 1987). Studies on the melanotropes of Xenopus luevis have suggested that POMC cells have yet another mechanism to modulate their biological signal. With pulse-chase experiments it was demonstrated that the cells release radiolabelled material in two phases (Martens et al., 1981). The first phase comprises a rapid increase in the amount of products released, reaching a maximum 3 hr after the pulse and a rapid decrease until 6 hr after the pulse; the second phase is characterized by a rather low but distinct release activity which remains constant for more than 12 hr.
These observations could mean that melanotropes possess two secretory compartments. The nature of the first phase (‘compartment’) has been established beyond doubt. High performance liquid chromatography (HPLC) studies have shown that it is associated with the release of newly synthesized POMC peptides (Martens et al., 1981). The second phase has not been analyzed. It might reflect release from the ‘constitutive pathway’ which appears to be a constant and unregulated pathway and is permanently involved in the delivery of membrane proteins to the plasma membrane in various cell types (for a review, see Burgess and Kelly, 1987). This pathway also accounts for the release of the intact precursor molecule, when sorting of secretory product in the Golgi apparatus is perturbed, as shown for a POMC-producing cell line (Gumbiner and Kelly, 1982) and for isolated mammalian islet p-cells (Burgess and Kelly, 1987). Alternatively, the second phase might represent regulated release from a ‘mature’ compartment containing POMC-derived peptides. The functioning of such a compartment in melanotropes would considerably add to the potential of these cells to generate and secrete diverse biological signals, particularly if this compartment would be regulated separately and/or would have a qualitatively different peptide content than the compartment associated with the first phase of release. The aim of the present study was to analyze release associated with the second phase, in order to determine whether it represents a regulated secretory compartment of POMC-derived peptides in Xenopus Zaevismelanotropes. First, using HPLC, it has been investigated whether the second phase of release of radiolabelled products from these cells reflects the release of POMC-derived peptides. Furthermore, to study whether the release during the second phase 199
INGRIDD. VAN Zmsr et al.
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Superfusion
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Time (Hours)
Fig. 1. Release of [?S]-methionine labelled material from neurointermediate lobes of Xenopus laeois in the absence (a) and presence (b) of 50 PM dopamine. Two phases in the release pattern are indicated. The results are from individual lobes; the experiments are repeated three times with very similar results. Those fractions that were included in HPLC analyses of peptide content (see Fig. 2) are indicated by the grey bars. reflects a constitutive (non-regulated) or a regulated pathway, the effect of dopamine on the release of radioactive material was assessed. Dopamine is known to be an inhibitor of the secretory activity of melanotrope cells of Xenopus luevis. Finally, to assess the ultimate fate of peptides during dopamineinduced inhibition, a dual pulse-label protocol was applied. This involved [3SS]-methionine to label dopamine-inhibited secretory material; following dopamine inhibition, [ 3H]-methionine labelling was conducted to distinguish between the release of newly synthesized and stored material. MATERIALS AND METHODS
Adult male Xenopus laeois were obtained from the Hubrecht Laboratory, Utrecht, The Netherlands, and kept in the laboratory on a grey background at a temperature of 22°C. At least three weeks prior to an experiment young adults (age: one year) were placed in black buckets to adapt to the background, under constant illumination. Experiment I. material-effect
Two phases of dopamine
of release
of radiolabelled
(0.14 M pyridine/0.5 M formic acid, pH 3.0) of this buffer to which 1% polypep (Sigma) had been added, 3 ml methanol and 6 ml buffer. After loading the column with a sample, it was washed with 3 ml 4% I-propanol in the buffer and eluted with 2 ml 40% I-propanol in the buffer. Four ml of scintillation fluid (Scintillator 199, Packard) was added to a 1 ml sample of the eluant and the amount of radioactivity determined in a liquid scintillation counter (LKB 1216 Rackbeta). The remainder of the sample was reserved for HPLC analysis of peptide content; samples for such analyses were pooled and evaporated to dryness (Speed-Vat concentrator, Savant). The column was regenerated for the next sample by washing with 3 ml methanol and 6 ml buffer. HPLC
Dried extracts were dissolved in the buffer and submitted to reversed-phase HPLC using a Spherisorb 10 ODS column (Bischoff, Leonberg) with 0.14 M pyridine/0.5 M formic acid as primary solvent and I-propanol as second solvent. Flow rate was 2ml/min (Martens et al., 1980). One ml fractions were mixed with scintillation fluid and their radioactivity measured in a liquid scintillation counter. Peaks were identified according to Martens et al. (1982a). Experiment 2. Dual labelling pulse-chase
analysis
Freshly dissected neurointermediate lobes were incubated for 45 min with 2.78 MBq [35S]-methionine (Amersham, specific activity: 2.9 TBq/mM) in sihconized glass vials, in 50 ~1 incubation medium, in a shaking water bath, at 22°C. The medium contained 112mM NaCl, 2mM KCI, 2mM CaCI,, 15 mM HEPES buffer (pH 7.4, carbon-aerated), 0.3 g/l bovine serum albumin (BSA, Sigma), 2 g/l glucose and 1mg/l ascorbic acid. Then the lobes were washed for I5 min in the medium, which contained 0.2 mM methionine (Calbiochem) and subsequently each lobe was placed individually in a superfusion chamber and superfused with the above medium, for 14 hr, with a flow rate of 1.5 ml/hr. Fractions of 30 min were collected in 100 ~1 0. I N HCI and stored at -20°C. In some experiments dopamine (50pM) was present in the superfusion medium.
Neurointermediate lobes were incubated for 45 min in medium containing 2.78 MBq [‘HI-methionine and washed for 15 min in medium with methionine as described in Experiment 1. Then, they were incubated for 5 hr in 50 p M dopamine-containing medium. This medium was refreshed every hour. Subsequently, the lobes were incubated for 45 min in medium containing 1.1 MBq [-‘?S]-methionine (Amersham, S.A. lOGBq/mM). Then the lobes were washed (15 min), and superfused with medium, for 14 hr. Samples, taken every 30 min, were extracted for peptide analysis as described in Experiment I and radioactivity associated with [3H]- and [35S]-methionine was determined.
Peptide estraction
Experiment
Fractions were submitted to chromatography to remove free radiolabelled amino acids, using a Baker octodecylC18 column. The column had been pretreated with 3 ml buffer
Following a pulse with [ 35S]-methionine, release of radiolabelled material from neurointermediate lobes was initially very low but showed a steep increase
RESULTS
I
Secretory
compartments
in neurointermediate
lobe of Xenopus laeuis
201
160 120 60 40
16 12 6 4
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HPLC Elution
35
40
45
50
Time (Minutes)
Fig. 2. HPLC profile [“S]-methionine labelled products in superfusion fractions of neurointermediate lobes of Xenopus laeuis, collected from 1.5 to 4.5 hr (top profile) and from 11 to 14 hr (lower profile) after labelling. Peaks are numbered according to Martens et al. (1982a), where I is a mixture of /I-MSH and y-MSH, II is desacetyl y-MSH, IV is a mixture of a-MSH and CLIP I, V is CLIP II, and VII and VIII represent peptides related to /I-endorphin. reaching a maximum after about 3 hr and decreased rapidly thereafter (Fig. la). This release, designated phase 1, was completed within about 6 hr. Thereafter release was at a moderate level, which persisted during the remainder of the experiment (second release phase). When dopamine was added to the chase medium, both phases of release were strongly inhibited (Fig. lb). HPLC analysis of the radioactivity released into the superfusion medium during the two phases (as indicated in Fig. 1) revealed the presence of a large number of peptides (Fig. 2). On the basis of retention times, c(-MSH, desacetyl cr-MSH, CLIP I, CLIP II and /l-endorphin-related peptides were identified. The total amount of peptide released during the first
phase was higher than that released during the second phase. However, the HPLC elution profile of peptides collected during the first release phase (from t = 1.5 to t = 4.5 hr) was similar to that of peptides collected during the second release phase (from t = 11 to t = 14 hr). Relative peak heights of some peptides, in particular the j?-endorphin-related peptides, differed between the phases, indicating differences in the relative amounts of peptide released. Experiment
2
Following the second pulse labelling (with [35S]methionine), both [ 3H]-labelled and [ 35S]-labe11ed products were present in the superfusion medium (Fig. 3). The pattern of release of [35S]-labelled
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Superfusion
Time
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Fig. 3. Release of [ ‘HI-methionine (A) and [ %I-methionine (m) labelled peptides from neurointermediate lobes of Xenopus laeuis. Lobes were first pulse-labelled with [‘HI-methionine followed by a 5 hr incubation in 50 PM dopamine, and then pulse-labelled with [‘%I-methionine. After superfusion, the amount of radiolabelled peptides in each 30 min superfusion fraction was determined. Results are expressed as a percentage of the total amount of radioactive peptides released. The data are the averages of six experiments; bars indicate standard errors of the mean.
202
INGRID D. VAN BEST
material was very similar to that found in Experiment 1, revealing two phases of release; during the first phase a clear peak (at t = 2.5 hr) was followed by a rapid decrease, whereas the second phase, starting at about t = 6 hr was characterized by a slowly declining release rate during the remainder of the chase period. The release of [3H]-labelled material, however, did not show a peak, but rather revealed a slowly declining release rate throughout the 14 hr chase period. From t = 6 hr onwards, this release pattern closely paralleled the release pattern of [ 3SS]-labelled material. DISCUSSION
The jirst
release phase
In the first experiment, two phases in the release of newly synthesized material from the neurointermediate lobe of Xenopus laevis have been observed. These phases are similar to those found by Martens ef al. (1981). The first phase lasts about 6 hr. The fact that it takes approximately 3 hr for the release in this phase to reach a maximum probably reflects the time needed for transport of newly synthesized material to the Golgi apparatus and for packing the material into secretory granules. The present HPLC data furthermore confirm the previous conclusion that the radioactive material released during the first phase represents newly synthesized peptides that are derived from POMC (Martens ef al., 1981). The present study shows that release of these peptides during this phase is clearly inhibited by dopamine. As dopamine is a potent inhibitor of the release of POMC peptides from the neurointermediate lobe of Xenopus laevis, it may well be that dopamine is involved in the in vivo control of first phase release from the lobe. The second
release phase
The identity and significance of the radioactive material released during the second phase have not been investigated before. The present HPLC study demonstrates that the material consists of the same POMC-derived peptides as released during the first phase. Apparently, the second phase reflects a regulated rather than a ‘constitutive’ pathway, because (I), processed end products are released (cf. Gumbiner and Kelly, 1982; Burgess and Kelly, 1987) and, particularly, (2) release can be completely inhibited by dopamine. Therefore, as in the case of the first release phase, dopamine may be involved in the control of second phase release of POMC-derived peptides from the Xenopus laevis neurointermediate lobe. Relation
between
two secretory
compartments
The above considerations permit the conclusion that there is a fast and a slow release compartment in the neurointermediate lobe of Xenopus faevis. The relation between these compartments appears from the results of Experiment 2, which showed that when release of peptides during the first phase (i.e. from the fast compartment) is prevented by dopamine, these peptides are shunted into the second (slow) release compartment; from here they are released when dopamine inhibition is terminated. Since the elution profiles obtained during the first
er al.
and second release phase are similar, it would seem that the peptide contents in the fast and slow compartments are similar as well. However, the observation might reflect the time constraint of our in vitro analysis: some steps in the processing of POMC are extremely slow and require much more time (some days) than the 14 hr of the experiment. This holds, for example, for the formation of C-terminally truncated forms of endorphin in mammals (Ham and Smith, 1984; Leenders et al., 1986) and, as to Xenopus laevis, for the formation of y -MSH by processing of the N-terminal region of POMC (Martens et al., 1982b). In view of the differences in peak height in the HPLC profiles, the possibility exists that the relative quantities of the individual peptides released differ between the two phases. Qualitative and/or quantitative differences between the peptide contents of the two compartments would considerably add to the multifunctional nature of the biological output of POMC-producing cells of the neurointermediate lobe in Xenopus laevis. With respect to the possible location of the two release compartments in the neurointermediate lobe, two possibilities could be envisaged. First, it has recently been shown that the pars intermedia of Xenopus laevis (de Rijk et al., 1990), as well as that of the rat (Back, 1989) contain two morphologically well-defined, functional stages of the melanotrope cell. Possibly, one cell stage represents the fast releasing compartment and the other the slowly releasing compartment. Alternatively, ultrastructural data suggest that the two compartments exist within the same cell, viz., in two types of secretory granules (Hopkins, 1970). Acknowledgements-The authors are greatly indebted to Mr B. W. M. M. Peeters and Mr P. M. J. M. Cruijsen for performing some of the experiments and to Mr R. J. C. Engels for animal care. This study was made possible by a grant from the Foundation for Biological Research (BION), which is subsidized by the Netherlands Organization for Scientific Research (NWO), and by a grant from the European Community (contract No. ST25-0468-C). REFERENCES
Back N. (1989) The effect of bromocryptine on the intermediate lobe.of the rat pituitary: an ejectron-microscopic, mornhometric studv. Cell Tiss. Res. 255, 405410. Burgess L. and Keily R. B. (1987) Constitutive and regulated secretion of proteins. Ann. Rev. Cell Biol. 3, 243-293. Eipper B. A. and Mains R. E. (1980) Structure and biosynthesis of pro-adrenocorticotropin/endorphin and related peptides. Endocr. Rev. 1, 1-27. Goldman M. E. and Loh Y. P. (1984) Intracellular acetylation of desacetyl-a-MSH in the Xenopus laevis neurointermediate lobe. Peptides 5, 112991134. Gumbiner B. and Kelly R. B. (1982) Two distinct intracellular pathways transport secretory and membrane glycoproteins to the surface of pituitary tumor cells. Cell 28, 51-59. Ham J., McFarthing K. G., Togood C. 1. A. and Smyth D. G. (1984) Influence of dopaminergic agents on /?endorphin processing in rat pars infermedia. Biochem. Sac. Trans. 12, 927-929. Ham J. and Smyth D. G. (1984) Regulation of bioactive /I-endorphin processing in rat pars infermedia. PEBS Lett. 175, 407411.
Secretory compartments
in neurointermediate
Herbert E. (1981) Discovery of proopiomelanocortin: a cellular polyprotein. Trends Biochem. Sci. 6, 184188. Hopkins C. R. (1970) Studies on secretory activity in the pars intermedia of Xenopus laevis. Tiss. Cell 2, 59-70. Leenders H. J., Janssens -J. J. W., Theunissen H. J. M., Jenks B. G. and Overbeeke A. P. van (1986) . , Acetvlation of melanocyte stimulating hormone and /?-endorphin in the pars intermedia of the perinatal pituitary gland in the mouse. Neuroendocrinol. 43, 166-174. Loh Y. P. and Jenks B. G. (1981) Evidence for two different turnover pools of adrenocorticotropin, a-melanocyte stimulating hormone, and endorphin-related peptides released by the frog pituitary neurointermediate lobe. Endocrinol. 109, 54-6 I.
Loh Y. P., Li A., Grietsch H. A. and Eskay R. L. (1981) Immunoreactive a-melanotropin and /?-endorphin in the toad pars intermedia; dissociation in storage, secretion and subcellular localization. Life Sci. 29, 1599-1605. Martens G. J. M., Jenks, B. G. and Overbeeke A. P. van (1980) Analysis of peptide biosynthesis in the neurointermediate lobe of Xenopus laevis using high performance liquid chromatography: occurrence of small bioactive products. Camp. Biochem. Physiol. 67B, 4933497.
Martens G. J. M., Jenks B. G. and Overbeeke A. P. van
lobe of Xenopus luevis
203
(1981) Microsuperfusion of neurointermediate lobes of Xenopus laevis: concomitant and coordinately controlled release of newly synthesized peptides. Comp. Biochem. Physiol. 69C, 75-82.
Martens G. J. M., Jenks B. G. and Overbeeke A. P. van (1982a) Biosynthesis of pairs of peptides related to melanotropin, corticotropin and endorphin in the pars intermedia of the amphibian pituitary gland. Eur. J. Biochem. 122, l-10. Martens G. J. M., Jenks B. G. and Overbeeke A. P. van (1982b) Biosynthesis of a y,-melanotropin-like peptide in the pars intermedia of the amphibian pituitary gland. Eur. J. Biochem. 126, 23-28.
Randle J. C. R., Moor B. C. and Kraicer J. (1983) Differential control of the release of pro-opiomelanocortin-derived peptides from the pars intermedia of the rat pituitary. Neuroendocrinol. 37, 131-140. Rijk E. P. C. T. de, Jenks B. G. and Wendelaar Bonga S. E. (1990) Morphology of the pars intermedia and the MSH-cells in Xenopus laevis in relation to background adaptation. Gen. Comp. Endocrinol. 78. Verburg-Van Kemenade B. M. L., Jenks B. G. and Smits R. J. M. (1987) N-terminal acetylation of MSH in thepars intermedia of Xenopus laevis is a physiologically regulated process. Neuroendocrinol. 46, 289-296.
0306~4492/90 $3.00+ 0.00 0 1990Pergamon Press plc
A SLOW AND A FAST SECRETORY COMPARTMENT OF POMC-DERIVED PEPTIDES IN THE NEUROINTERMEDIATE LOBE OF THE AMPHIBIAN XENOPUS INGRID D. VAN ZOEST, HANS J.
LAEVIS
LEENDERS, BRUCEG. JENKSand ERIC W. ROUBOS
Department of Animal Physiology, Faculty of Science, Catholic University of Nijmegen, Toernooiveld, 6525 ED Nijmegen, The Netherlands. Telephone: (080) 652036, Fax: (080) 553450 (Received 24 January 1990)
Peptide release from the neurointermediate lobe of Xenopus lueuishas been studied using dual pulse-chase incubation, superfusion and HPLC techniques. 2. Lobes release pulse-labelled material in two phases, the first phase lasting about 6 hr, the second persisting up to 14 hr. 3. In both phases similar, POMC-derived peptides are released. Their release can be inhibited by dopamine. 4. When release during the first phase is inhibited, newly synthesized peptides are shunted into the second release pathway. 5. It is concluded that the neurointermediate lobe contains two release compartments. The possible locations of these compartments within melanotrope cells have been discussed. Abstract-l.
INTRODUCTION The biological action of endocrine cells producing the precursor protein proopiomelanocortin (POMC) is determined by the mode of processing of POMC and by the dynamics of the release of POMC-derived peptides. The direction of POMC processing is largely cell-specific. For example, corticotrope cells of the pars distalis of the pituitary gland produce ACTH as a major secretory product, whereas in the melanotrope cells of the pars intermedia, ACTH is further processed to form cr-MSH as a secretory product (Eipper and Mains, 1980; Herbert, 1981). However, within a given cell type, the mode of POMC processing may be, to a certain extent, open to modulation. Much of the evidence for this comes from in vitro experiments, in which POMC cells were treated with pharmacological agents (Loh and Jenks, 1981; Loh et al., 1981; Randle et al., 1983; Goldman and Loh, 1984; Ham and Smyth, 1984; Ham et al., 1984), but in the case of the melanotropes of the South African clawed toad Xenopus laevis it has been shown that a physiological stimulus (background adaptation) can alter POMC processing and, hence, the in vivo profile of the released peptides (Verberg-van Kemenade et al., 1987). Studies on the melanotropes of Xenopus luevis have suggested that POMC cells have yet another mechanism to modulate their biological signal. With pulse-chase experiments it was demonstrated that the cells release radiolabelled material in two phases (Martens et al., 1981). The first phase comprises a rapid increase in the amount of products released, reaching a maximum 3 hr after the pulse and a rapid decrease until 6 hr after the pulse; the second phase is characterized by a rather low but distinct release activity which remains constant for more than 12 hr.
These observations could mean that melanotropes possess two secretory compartments. The nature of the first phase (‘compartment’) has been established beyond doubt. High performance liquid chromatography (HPLC) studies have shown that it is associated with the release of newly synthesized POMC peptides (Martens et al., 1981). The second phase has not been analyzed. It might reflect release from the ‘constitutive pathway’ which appears to be a constant and unregulated pathway and is permanently involved in the delivery of membrane proteins to the plasma membrane in various cell types (for a review, see Burgess and Kelly, 1987). This pathway also accounts for the release of the intact precursor molecule, when sorting of secretory product in the Golgi apparatus is perturbed, as shown for a POMC-producing cell line (Gumbiner and Kelly, 1982) and for isolated mammalian islet p-cells (Burgess and Kelly, 1987). Alternatively, the second phase might represent regulated release from a ‘mature’ compartment containing POMC-derived peptides. The functioning of such a compartment in melanotropes would considerably add to the potential of these cells to generate and secrete diverse biological signals, particularly if this compartment would be regulated separately and/or would have a qualitatively different peptide content than the compartment associated with the first phase of release. The aim of the present study was to analyze release associated with the second phase, in order to determine whether it represents a regulated secretory compartment of POMC-derived peptides in Xenopus Zaevismelanotropes. First, using HPLC, it has been investigated whether the second phase of release of radiolabelled products from these cells reflects the release of POMC-derived peptides. Furthermore, to study whether the release during the second phase 199
INGRIDD. VAN Zmsr et al.
200
4
2
6
Superfusion
6
10
12
14
Time (Hours)
Fig. 1. Release of [?S]-methionine labelled material from neurointermediate lobes of Xenopus laeois in the absence (a) and presence (b) of 50 PM dopamine. Two phases in the release pattern are indicated. The results are from individual lobes; the experiments are repeated three times with very similar results. Those fractions that were included in HPLC analyses of peptide content (see Fig. 2) are indicated by the grey bars. reflects a constitutive (non-regulated) or a regulated pathway, the effect of dopamine on the release of radioactive material was assessed. Dopamine is known to be an inhibitor of the secretory activity of melanotrope cells of Xenopus luevis. Finally, to assess the ultimate fate of peptides during dopamineinduced inhibition, a dual pulse-label protocol was applied. This involved [3SS]-methionine to label dopamine-inhibited secretory material; following dopamine inhibition, [ 3H]-methionine labelling was conducted to distinguish between the release of newly synthesized and stored material. MATERIALS AND METHODS
Adult male Xenopus laeois were obtained from the Hubrecht Laboratory, Utrecht, The Netherlands, and kept in the laboratory on a grey background at a temperature of 22°C. At least three weeks prior to an experiment young adults (age: one year) were placed in black buckets to adapt to the background, under constant illumination. Experiment I. material-effect
Two phases of dopamine
of release
of radiolabelled
(0.14 M pyridine/0.5 M formic acid, pH 3.0) of this buffer to which 1% polypep (Sigma) had been added, 3 ml methanol and 6 ml buffer. After loading the column with a sample, it was washed with 3 ml 4% I-propanol in the buffer and eluted with 2 ml 40% I-propanol in the buffer. Four ml of scintillation fluid (Scintillator 199, Packard) was added to a 1 ml sample of the eluant and the amount of radioactivity determined in a liquid scintillation counter (LKB 1216 Rackbeta). The remainder of the sample was reserved for HPLC analysis of peptide content; samples for such analyses were pooled and evaporated to dryness (Speed-Vat concentrator, Savant). The column was regenerated for the next sample by washing with 3 ml methanol and 6 ml buffer. HPLC
Dried extracts were dissolved in the buffer and submitted to reversed-phase HPLC using a Spherisorb 10 ODS column (Bischoff, Leonberg) with 0.14 M pyridine/0.5 M formic acid as primary solvent and I-propanol as second solvent. Flow rate was 2ml/min (Martens et al., 1980). One ml fractions were mixed with scintillation fluid and their radioactivity measured in a liquid scintillation counter. Peaks were identified according to Martens et al. (1982a). Experiment 2. Dual labelling pulse-chase
analysis
Freshly dissected neurointermediate lobes were incubated for 45 min with 2.78 MBq [35S]-methionine (Amersham, specific activity: 2.9 TBq/mM) in sihconized glass vials, in 50 ~1 incubation medium, in a shaking water bath, at 22°C. The medium contained 112mM NaCl, 2mM KCI, 2mM CaCI,, 15 mM HEPES buffer (pH 7.4, carbon-aerated), 0.3 g/l bovine serum albumin (BSA, Sigma), 2 g/l glucose and 1mg/l ascorbic acid. Then the lobes were washed for I5 min in the medium, which contained 0.2 mM methionine (Calbiochem) and subsequently each lobe was placed individually in a superfusion chamber and superfused with the above medium, for 14 hr, with a flow rate of 1.5 ml/hr. Fractions of 30 min were collected in 100 ~1 0. I N HCI and stored at -20°C. In some experiments dopamine (50pM) was present in the superfusion medium.
Neurointermediate lobes were incubated for 45 min in medium containing 2.78 MBq [‘HI-methionine and washed for 15 min in medium with methionine as described in Experiment 1. Then, they were incubated for 5 hr in 50 p M dopamine-containing medium. This medium was refreshed every hour. Subsequently, the lobes were incubated for 45 min in medium containing 1.1 MBq [-‘?S]-methionine (Amersham, S.A. lOGBq/mM). Then the lobes were washed (15 min), and superfused with medium, for 14 hr. Samples, taken every 30 min, were extracted for peptide analysis as described in Experiment I and radioactivity associated with [3H]- and [35S]-methionine was determined.
Peptide estraction
Experiment
Fractions were submitted to chromatography to remove free radiolabelled amino acids, using a Baker octodecylC18 column. The column had been pretreated with 3 ml buffer
Following a pulse with [ 35S]-methionine, release of radiolabelled material from neurointermediate lobes was initially very low but showed a steep increase
RESULTS
I
Secretory
compartments
in neurointermediate
lobe of Xenopus laeuis
201
160 120 60 40
16 12 6 4
5
10
15
20
30
25
HPLC Elution
35
40
45
50
Time (Minutes)
Fig. 2. HPLC profile [“S]-methionine labelled products in superfusion fractions of neurointermediate lobes of Xenopus laeuis, collected from 1.5 to 4.5 hr (top profile) and from 11 to 14 hr (lower profile) after labelling. Peaks are numbered according to Martens et al. (1982a), where I is a mixture of /I-MSH and y-MSH, II is desacetyl y-MSH, IV is a mixture of a-MSH and CLIP I, V is CLIP II, and VII and VIII represent peptides related to /I-endorphin. reaching a maximum after about 3 hr and decreased rapidly thereafter (Fig. la). This release, designated phase 1, was completed within about 6 hr. Thereafter release was at a moderate level, which persisted during the remainder of the experiment (second release phase). When dopamine was added to the chase medium, both phases of release were strongly inhibited (Fig. lb). HPLC analysis of the radioactivity released into the superfusion medium during the two phases (as indicated in Fig. 1) revealed the presence of a large number of peptides (Fig. 2). On the basis of retention times, c(-MSH, desacetyl cr-MSH, CLIP I, CLIP II and /l-endorphin-related peptides were identified. The total amount of peptide released during the first
phase was higher than that released during the second phase. However, the HPLC elution profile of peptides collected during the first release phase (from t = 1.5 to t = 4.5 hr) was similar to that of peptides collected during the second release phase (from t = 11 to t = 14 hr). Relative peak heights of some peptides, in particular the j?-endorphin-related peptides, differed between the phases, indicating differences in the relative amounts of peptide released. Experiment
2
Following the second pulse labelling (with [35S]methionine), both [ 3H]-labelled and [ 35S]-labe11ed products were present in the superfusion medium (Fig. 3). The pattern of release of [35S]-labelled
I
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I
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I
2
4
6
6
10
12
14
Superfusion
Time
(Hours)
Fig. 3. Release of [ ‘HI-methionine (A) and [ %I-methionine (m) labelled peptides from neurointermediate lobes of Xenopus laeuis. Lobes were first pulse-labelled with [‘HI-methionine followed by a 5 hr incubation in 50 PM dopamine, and then pulse-labelled with [‘%I-methionine. After superfusion, the amount of radiolabelled peptides in each 30 min superfusion fraction was determined. Results are expressed as a percentage of the total amount of radioactive peptides released. The data are the averages of six experiments; bars indicate standard errors of the mean.
202
INGRID D. VAN BEST
material was very similar to that found in Experiment 1, revealing two phases of release; during the first phase a clear peak (at t = 2.5 hr) was followed by a rapid decrease, whereas the second phase, starting at about t = 6 hr was characterized by a slowly declining release rate during the remainder of the chase period. The release of [3H]-labelled material, however, did not show a peak, but rather revealed a slowly declining release rate throughout the 14 hr chase period. From t = 6 hr onwards, this release pattern closely paralleled the release pattern of [ 3SS]-labelled material. DISCUSSION
The jirst
release phase
In the first experiment, two phases in the release of newly synthesized material from the neurointermediate lobe of Xenopus laevis have been observed. These phases are similar to those found by Martens ef al. (1981). The first phase lasts about 6 hr. The fact that it takes approximately 3 hr for the release in this phase to reach a maximum probably reflects the time needed for transport of newly synthesized material to the Golgi apparatus and for packing the material into secretory granules. The present HPLC data furthermore confirm the previous conclusion that the radioactive material released during the first phase represents newly synthesized peptides that are derived from POMC (Martens ef al., 1981). The present study shows that release of these peptides during this phase is clearly inhibited by dopamine. As dopamine is a potent inhibitor of the release of POMC peptides from the neurointermediate lobe of Xenopus laevis, it may well be that dopamine is involved in the in vivo control of first phase release from the lobe. The second
release phase
The identity and significance of the radioactive material released during the second phase have not been investigated before. The present HPLC study demonstrates that the material consists of the same POMC-derived peptides as released during the first phase. Apparently, the second phase reflects a regulated rather than a ‘constitutive’ pathway, because (I), processed end products are released (cf. Gumbiner and Kelly, 1982; Burgess and Kelly, 1987) and, particularly, (2) release can be completely inhibited by dopamine. Therefore, as in the case of the first release phase, dopamine may be involved in the control of second phase release of POMC-derived peptides from the Xenopus laevis neurointermediate lobe. Relation
between
two secretory
compartments
The above considerations permit the conclusion that there is a fast and a slow release compartment in the neurointermediate lobe of Xenopus faevis. The relation between these compartments appears from the results of Experiment 2, which showed that when release of peptides during the first phase (i.e. from the fast compartment) is prevented by dopamine, these peptides are shunted into the second (slow) release compartment; from here they are released when dopamine inhibition is terminated. Since the elution profiles obtained during the first
er al.
and second release phase are similar, it would seem that the peptide contents in the fast and slow compartments are similar as well. However, the observation might reflect the time constraint of our in vitro analysis: some steps in the processing of POMC are extremely slow and require much more time (some days) than the 14 hr of the experiment. This holds, for example, for the formation of C-terminally truncated forms of endorphin in mammals (Ham and Smith, 1984; Leenders et al., 1986) and, as to Xenopus laevis, for the formation of y -MSH by processing of the N-terminal region of POMC (Martens et al., 1982b). In view of the differences in peak height in the HPLC profiles, the possibility exists that the relative quantities of the individual peptides released differ between the two phases. Qualitative and/or quantitative differences between the peptide contents of the two compartments would considerably add to the multifunctional nature of the biological output of POMC-producing cells of the neurointermediate lobe in Xenopus laevis. With respect to the possible location of the two release compartments in the neurointermediate lobe, two possibilities could be envisaged. First, it has recently been shown that the pars intermedia of Xenopus laevis (de Rijk et al., 1990), as well as that of the rat (Back, 1989) contain two morphologically well-defined, functional stages of the melanotrope cell. Possibly, one cell stage represents the fast releasing compartment and the other the slowly releasing compartment. Alternatively, ultrastructural data suggest that the two compartments exist within the same cell, viz., in two types of secretory granules (Hopkins, 1970). Acknowledgements-The authors are greatly indebted to Mr B. W. M. M. Peeters and Mr P. M. J. M. Cruijsen for performing some of the experiments and to Mr R. J. C. Engels for animal care. This study was made possible by a grant from the Foundation for Biological Research (BION), which is subsidized by the Netherlands Organization for Scientific Research (NWO), and by a grant from the European Community (contract No. ST25-0468-C). REFERENCES
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