Synthesis of multiple peptides from a larger precursor in the neuroendocrine caudo-dorsal cells of Lymnaea stagnalis

Neuroscience Letters, 56 (1985) 241 246

241

Elsevier Scientific Publishers Ireland Ltd.

NSL 03305

S Y N T H E S I S OF M U L T I P L E P E P T I D E S F R O M A LARGER P R E C U R S O R IN T H E N E U R O E N D O C R I N E C A U D O - D O R S A L CELLS OF

L YMNAEA S T A G N A L I S

W.P.M. GERAERTS*, E. V R E U G D E N H I L , R.H.M. EBBERINK and Th.M. HOGENES

Biological Laboratory, Vrije Universiteit, Postbox 7161, IO07MC Amsterdam (The Netherlands) (Received November 12th, 1984; Revised version received January 25th, 1985; Accepted February 28th, 1985)

Key word~: peptidergic cells- percursor processing axonal transport

multiple peptide release - snail

The biosynthesis, transport and release of multiple peptides by the egg-laying controlling neuroendocrine caudo-dorsal cells (CDCs) of Lymnaea stagnalis were studied. High-performance gel permeation chromatography was used to resolve newly synthesized peptides after pulse-chase experiments with radioactive amino acids. The ultimate precursor is a ~ 35 kd (K) peptide which is produced in the CDC somata. It gives rise to intermediate products ( ~ 2 0 K, ~ 10 K and ~ 7 K) and a number of end products which include a ~4.5 K peptide (the ovulation hormone) and other peptides ( ~ 6 K, ~3.5 K and ~ 2 K). The end products are transported in neurosecretory granules to the~CDC axon terminals in the cerebral commissure where they are released into the medium during electrical discharges of the CDC system.

Egg-laying in the freshwater snail Lymnaea stagnalis is controlled by the caudodorsal cells (CDCs), bilateral clusters of neuroendocrine cells located in the cerebral ganglia [3, 11]. In isolated brains, repeated intracellular stimulation of a single CDC triggers a 50-min burst of synchronous spiking activity (discharge) in all 100 CDCs [9]. During a discharge the cells release various peptides from the contents of neurosecretory granules in axon terminals located at the periphery of the intercerebral commissure (COM) [4-6, 9, 1i]. Egg-laying is characterized by a stereotyped pattern of behaviours which involve ovulation of numerous oocytes, egg formation, egg mass formation, cessation of locomotion, turning of the body, alterations in feeding movements and oviposition [2, 8]. These events are thought to be initiated and co-ordinated by the neuropeptides that are released during the discharge [4, 6, 8]. The most extensively studied is the ovulation hormone (CDCH), a basic peptide with a Mr of "~4.7 kdalton (K) [7]. It affects identified neurones in the neuronal feeding network besides inducing egg mass production [8]. In vivo, a CDC discharge is accompanied by a massive release of CDCH into the blood which always precedes egg mass production. The function of the other peptides in egg-laying is as yet unknown.

*Author for correspondence. 0304-3940/85/$ 03.30 © 1985 Elsevier Scientific Publishers Ireland Ltd.

3

o

I

o

-

o------O ~ l

-

I

,

~

I

o

I

*7

I

~

O

1

,

~

~

- -

,

O_

T

°

. ~

,

•~

~ ' ~

i

I-

f

aND

- - - - - ~

,

,

~~

o-

~

°

I ~ -

~

-

~

T

O~O

T

~

~

°

•

~

~ ~-

T

-

°

.

-lifo

j

~

O

°'e°-~P

~

•

- -

•

"~

--O--

•

I

-o

c

o

i

•

cpm I~ 2

cpm I0 -z

z

u~ o ~u

~o

z

c--~ t-tl c~o

i

243

The co-ordinate release of multiple peptides raises the question of their mode of synthesis and transport. This has been studied in only few invertebrate neuro-endocrine systems, including the egg-laying controlling bag cells of the marine snail Aplysia californica [l, 13-15, 17, 18]. The CDC system provides a valuable preparation for such studies because (1) the CDC somata and their axon terminals are separated in anatomically discrete regions [l 1], (2) the CDC clusters consist of large (ca. 70 #m diameter) homogeneous neuroendocrine cells and the axon terminals at the periphery of the COM are almost exclusively of the CDC type [1 l] and (3) the CDC system can be induced to secrete CDCH and other peptides when kept for extended periods in vitro [4]. Here we wish to present circumstantial evidence that the CDC secretory peptides are derived from (a) larger precursor molecule(s) and are transported intragranularly to the CDC axon terminals where they are released during bursts of electrical activity of the CDC system. Pairs of cerebral ganglia, connected by the COM, were dissected from adult laboratory raised Lymnaea stagnalis; all nerves were cut at the origin. These preparations are referred to as CDC/COMs. They were collected in sterilized snail Ringer kept at pH ~ 7.8 with 1.73% in 02. CDC/COMs, incubated in groups of 20, were given a 10-min pulse incubation at 20°C in 250 #i fresh Ringer (pH ~ 7.8) containing 100/~Ci of a mixture of 15 tritiated amino acids (TRK 440; Amersham). One group of CDC/COMs was then processed for high-performance gel permeation chromatography (HPGPC). The other groups were first rinsed for 15 rain in 3 successive l-ml rinses of fresh Ringer containing 1 mM unlabelled amino acids and then chase-incubated in this solution for various lengths of time. The incubations were terminated by placing the incubation vials on ice and adding cold Ringer in excess. Isolation of the CDC somata and the COM was carried out under cold Ringer to which the protease inhibitor aprotinin (Sigma) was added in a concentration of 1 trypsin inhibitor unit/ml Ringer [4]. The transparent perineurium surrounding the cerebral ganglia was opened above the CDCs, and the clusters o f C D C somata were eased out. The central third part of the COM was then dissected free from other tissues. Homogenization was carried out in HPGPC eluent solvent. After centrifugation (5 min, 6000 g) the supernatants were stored frozen until further analysis. In experiments on labelled release, the CDC/COMs were first pulse-chase incubated and then placed in fresh Ringer (250/tl) containing aprotinin but without the amino acids (control solution). After 1 h this solution was replaced with 250 ~tl fresh Ringer with aprotinin and with 2 mM 8-cpt-cAMP [8-(4-chlorophenylthio)ade-

Fig. 1. High-performance gel permeation radiochromatograms of newly synthesized peptides in the C D C system of Lymnaea stagnalis. C D C / C O M s were pulse-labelled for 10 min in Ringer containing a mixture of 15 tritiated amino acids and chased in non-radioactive Ringer as described in the text. The data refer to one C D C system, i.e. 100 C D C s and one entire C O M (3 × 1/3 COM), Because no radioactive material was eluted during the first 25 min, the first part of the cbromatogram is not shown. Markers run concurrently are: Blue Dextran 200 (mol.wt. 2 × 106), ovalbumin (43,000), chymotrypsin (25,000), ribonuclease (13,700). glucagon (3500), bacitracin (1450) and leucine (131 ).

244

nosine-3',5'-monophosphate] to induce electrical activity and concomittant release [4, 11] (stimulation solution). The solutions were taken up in HPGPC eluent solvent and stored frozen until HPGPC analysis. The HPGPC instrumentation (Waters) consisted of a pump (model M45) and an injector (model U6K). Further instrumentation used consisted of an LKB 2138 Uvicord S with HPGPC flow cell and a Pharmacia Fracl00 fraction collector. Aliquots of 100-250 y1 to which peptide markers were added were injected on the following combination of Protein-Pak columns (each 0.78 cm i.d. x 30 cm; Waters) in series, namely an 1-60, two 1-125 and an 1-300 column in this order [16]. This combination separates molecules in the range of 1.5 to 90K predominantly by size. The columns were eluted with a mixture of 4 M guanidine-hydrochloride and 0.2 M triethylamine (pH 3.0). The flow-rate was 1 ml/min and fractions of 250 /tl (15 s) were collected in minivials. The absorbance of the peptide markers was monitored at 280 nm. For the determination of radioactivity each column fraction was added to 5 ml of Dynagel (Baker) and counted for 10 min (Mark III). All data have been corrected for background and quenching. Pulse-chase analysis demonstrated the biosynthesis of at least 8 peptides (Fig. 1) corresponding to Mrs of ~ 3 5 K, ~ 2 0 K, ~ 10 K, ~ 7 K, ~ 6 K, ~4.5 K, ~3.5 K and ~ 2 K, referred to here as peaks l-VIII. The labelled protein peaks with Mrs of > 40 K have different turnover rates and are apparently unrelated to the C D C H processing sequence. After a 10-min pulse of labelled amino acids, peak I was very prominent in the C D C somata, whereas peak II was small. After 2 h ofchase-incuba-

AXON M W 10-3

a3

RINI]ER I I [ D [ system )

TERMINALS

I I COM

]

25

13 7

35

i

i

i

1 L.5 0 13 I

MWI0 J

i

20 h

/.3

25

13 7

35

I

I

I

l

l.&5 0 13 I

I

release

~8-

chase o--e control o--o sfimulafed [with cAMP}

_

L

25

0

35

~5 TIME Iminl

25

35

t,S TIHE Iminl

Fig. 2. High-performance gel permeation radiochromatograms of tritiated peptides synthesized, stored and released by the C D C system of Lymnaea stagnalis. CDC/COM preparations were pulseqabelled for 10 rain in Ringer containing a mixture of 15 radioactive amino acids and chased in non-radioactive Ringer for 24 h. For further details, see text and Fig. 1. A: radiochromatogram of tritiated peptides from one entire COM (3 x 1/3 COM). B: radiochromatograms of control and stimulation solutions. The data refer to releasc from one CDC system. Release was stimulated with 8-cpl-cAMP.

245 tion, peak I had considerably decreased, while peak II had increased. Also other peaks (III-VI) were present. Thereafter the level of peak II decreased gradually. Concommittantly peaks VII and VIII appeared, and between 2 and 8 h of chase-incubation peaks V-VIII also appeared in the COM. Similar results were obtained with preparations that were preincubated for 24 h in Ringer. This shows that the CDCs of the preparations function normally, even after prolonged periods in vitro. The results strongly suggest that in the CDCs the co-ordinate production of peptides proceeds through a multi-step proteolytic processing sequence analogous to those which have been demonstrated for peptides in other systems, including the bag cells of A. californica [1, 10, 12-15]. In L. stagnalis the precursor very likely is the 35 K peptide which is produced in the CDC somata. One must keep in mind, however, that HPGPC separates molecules on the basis of molecular size. Hence we are dealing with molecular weight classes and not necessarily with distinct peptides. The possibility that the ~ 35 K region contains multiple precursors is under investigation. The precursor(s) give(s) rise to intermediate products which include peptides with Mrs of "~20 K, "-~10 K and ~7 K. Because these intermediates do not appear in the CDC axon terminals of the COM parts dissected, we must conclude that their further processing is confined to the CDC somata and perhaps the proximal parts of the CDC axons (which were not studied in the present experiments). The end products include peptides with Mrs of ~6 K, ~4.5 K (probably CDCH), ~3.5 K and 2 K. The fact that these peptides appear in the COM only after several hours of chase-incubation indicates that they are transported from the CDC somata to the axon terminal. Several lines of evidence suggest that this transport occurs intragranularly. In previous studies we have shown that ovulation hormone activity is mainly associated with purified CDC neurosecretory granules [5]. Light and electron microscopic autoradiography shows that the 14C-labelled material transported to the COM is in the CDC neurosecretory granules (E.W. Roubos, personal communication). Also, under conditions where axonal transport of the granules is blocked with vinblastine, accumulation of labelled intermediates and end-products occurs in the CDC somata, while almost no end-products appear in the COM (unpublished results). The experiments on labelled release show that during the control period a nonstimulus-dependent background leakage occurred of higher (Mr> 40 K) and lower (< 1.5 K) molecular weight material from the preparations into the medium (Fig. 2). After stimulation with 8-cpt-cAMP, the electrically active CDC system released radioactive peptides that co-eluted with the peaks V-VIII present in COM extracts. (There was also increase of the Mr>40 K and < 1.5 K material.) This strongly suggests that it is the contents of neurosecretory granules in the CDC axon terminals that have been released. The total of the Mrs of the end-products is not equal to the Mr of the precursor. Also in this case it is possible, however, that each peak of radioactive material is in fact composed of several different peptides. We thank Professor J. Joosse for critical reading of the manuscript, Dr. B.G. Jenks for helpful discussion and Thea Laan for typing of the manuscript.

246

1 Berry, R.W., Proteolytic processing in the biogenesis of the neurosecretory eggqaying hormone in dplysia. 1. Precursors, intermediates and products, Biochemistry, 20 (1981) 6200-6205. 2 Dogterom, G.E., Bohlken, S. and Joosse, J., Effect of the photoperiod on the time schedule of egg mass production in Lymnaea slagnali& as induced by ovulation hormone injections, Gen. Comp. Endocrinol., 49 (1983) 255 260. 3 Geraerts, W.P.M. and Bohlken, S., The control of ovulation in the hermaphrodite freshwater snail l, rmnaea stagnalis by the neurohormone of the caudodorsal cells, Gen. Comp. Endocrinol., 28 (t976) 350 357. 4 Geraerts, W.P.M. and Hogenes, Th.M., Heterogeneity of peptides released by electrically active neuroendocrine caudo-dorsal cells of Lyrnnaea slagnalis, Brain Res,, in press. 5 Geraerts, W.P.M., Buma, P. and Hogenes, Th.M., Isolation of neurosecretory granules from the neurohaemal areas of peptidergic systems of Lyrnnaea stagnalis, with special reference to the ovaluation hormone-producing caudodorsal cells, Gen. C0mp. Endocrinol., 53 (1984) 212 217. 6 Geraerts, W.P.M., Tensen, C. and Hogenes, Th.M., Multiple release of peptides by electrically active neurosecretory caudo-dorsal cells of Lymnaea slagnalis, Neurosci. Lett., 41 (1983) 151 155. 7 Geraerts, W.P.M., Cheeseman, P., Ebberink, R.H,M., Nuyt, K. and Hogenes, Th.M., Partial purification and characterization of the ovulation hormone of the freshwater pulmonate snail Lymnaea staghalls, Gen. Comp. Endocrinol., 51 (1983)471-~76. 8 Goldschmeding, J.T., Wilbrink, M. and ter Maat, A., The role of the ovulation hormone in the control of egg-laying behaviour in L rrnnaea stagnalis, In J. Lever and H,H. Boer (Eds.), Molluscan Neuroendocrinology, Elsevier/North Holland, Amsterdam, Oxford, New York, 1983, pp. 251--256. 9 Kits, K.S., States of excitability in ovulation hormone producing neuro-endocrine cells of L)'mnaea slagnalis (Gaslropoda) and their relation to the egg-laying cycle, J. Neurobiol., I 1 (1980) 397 410. l0 Loh, Y.P. and Chang, T.-L., Pro-opiocortin converting activity in rat intermediate and neural lobe secretory granules, FEBS Lett., 137 (1982) 57 62. 11 Roubos, E,W., Cytobiology of the ovulation hormone producing neuroendocrine caudo-dorsal cells of L rmnaea stagnalis, Int. Rev. Cytol., 89 (1984) 295 346. 12 Russell, J.T., Brownstein, M.J. and Gainer, H., Time courses of appearance and release of [35S]cysteine labelled neurophysins and peptides in the neurohypophysis, Brain Res., 205 (1981) 299 311. 13 Scheller, R.It., Jackson, J.F., McAllister, L.B., Schwartz, J.H., Kandel, E.R. and Axel, R., A family of genes that codes for ELI{, a neuropeptide eliciting a stereotyped pattern of behaviour in Aplvsia. Cell, 28 (1982) 707 719. 14 Scheller, R.H., Rothman, B.S. and Mayeri, R., A single gene encodes multiple peptide transmitter candidales involved in a stereotyped behavior, TINS, 6 (1983) 340 345. 15 Scheller, R.H., Jackson, J.F., McAIlister, L.B., Rothman, B.S., Mayeri, E. and Axel, R., A single gent encodes multiple neuropeptides mediating a stereotyped behavior, Cell, 32 (1982) 7 22. 16 Seidah, N.G. and Chr6tien, M., Application of high-performance liquid chromatography to characterize radiolabelled peptides for radioimmunoassay, biosynthesis and microsequence studies of polypeptide hormones. In J.J, Langone and H. Van Vunakis (Eds.), Methods in Enzymology, Vol. 92, lmmunocytochemical Techniques, Part E, Monoclonal Antibodies and General Immunoassay Methods. Academic Press, New York, 1983, pp. 292 309. 17 Stuart, D.K., Chiu, A.Y. and Strumwasser, F., Neurosecretion of egg-laying hormone and other peptides from electrically active bag cell neurones of Aplysia, J. Neurophysiol., 43 (1980) 488 498. 18 Stuenkel, E.L., Biosynthesis and axonal transport of proteins and identified peptide hormones in the X-organ sinus gland neurosecretory system, J. Comp. Physiol., B153 (1983) 191 205.