Quantitative determination and distribution of the myotropic neuropeptide orcokinin in the nervous system of astacidean crustaceans

Peptides, Vol. 15, No. 3, pp. 393-400, 1994 Copyright © 1994 ElsevierScience Ltd Printed in the USA. All rights reserved 0196-9781/94 $6.00 + .00

Pergamon 0196-9781 (93)E0036-Q

Quantitative Determination and Distribution of the Myotropic Neuropeptide Orcokinin in the Nervous System of Astacidean Crustaceans DIETER

BUNGART, l HEINRICH

DIRCKSEN

AND

RAINER

KELLER

Institut ffir Zoophysiologie, R h e i n i s c h e Friedrich- W i l h e l m s - Universita't, E n d e n i c h e r Allee 11-13, D-53115 Bonn, G e r m a n y R e c e i v e d 23 A u g u s t 1993 BUNGART, D., H. DIRCKSEN AND R. KELLER. Quantitative determination and distribution of the myotropic neuropeptide orcokinin in the nervous system of astacidean crustaceans. PEPT1DES 15(3) 393-400, 1994.--For quantitative determinations of orcokinin, an indirect, noncompetitive sandwich ELISA was developed. This ELISA is highly specific for orcokinin and the detection limit is I fmol. In three astacidean species (Orconectes limosus, Homarus americanus, and Astacus astacus) orcokinin immunoreactivity (OK-IR) was measurable in all parts of the nervous system. Upon normalization to the protein content of the tissue (pmol/mg protein), concentrations were shown to be in the same range in all three species. The distribution of OK-IR in the nervous system is also very similar in the three species. In Orconectes limosus the following values were obtained (in pmol/ mg protein): cerebral ganglion 215, optic ganglia in the eyestalk 38, subesophageal ganglion 182. The thoracic ganglia have lower concentrations (35-72) and the abdominal ganglia (AG) 1-5 even lower ones (11-17). In the AG 6 of Orconectes, from which the innervation of the hindgut arises, concentrations are approximately five times higher than in the other AG. In hindgut tissue, relatively high concentrations of 22 pmol/mg were measured, which is in agreement with the demonstrated function of orcokinin as a hindgut excitatory substance. Markedly elevated levels of orcokinin were observed in the AG 6 of Astacus, but not in Homarus. Orcokinin could also be measured consistently and reliably in the hemolymph, where its concentration is approximately 1 × 10-~z M. These results show that orcokinin may be released into the hemolymph and may act as a hormone, in addition to its role as a locally acting neurotransmitter/modulator. Crustacea Orconecteslimosus Astacus astacus Neuropeptide Enzyme immunoassay

Homarus americanus

Myotropic peptide

Orcokinin

contraction was found to be three to five times higher than that ofproctolin, FLI4 (a FaRP peptide) (29), and CCAP, and reached a m a x i m u m of about 1000% increase at a concentration of I X I0 7 M. The threshold concentration was approximately 1 × 10 l ° M . With regard to the distribution and physiology of orcokinin, we are still at a very preliminary stage. There is as yet no information on its distribution in the nervous system and occurrence in crustacean species other than Orconectes limosus or in other invertebrate groups. To provide the requisite tool for this and related studies, we have developed an enzyme linked i m m u nosorbent assay (ELISA), based on a polyclonal antiserum to orcokinin. By means of this ELISA, we were able to determine quantitatively the distribution oforcokinin in the nervous system of Orconectes limosus. Furthermore, the occurrence and distribution of an orcokinin-immunoreactive substance in two other astacidean species, the lobster Hornarus americanus and the crayfish Astacus astacus, will be reported.

IN recent years, a large number of myotropic and cardioactive peptides have been isolated from nervous tissues of various invertebrates, particularly molluscs and insects [for reviews see (8,15,17,19)]. In comparison, the number of identified cardioactive/myotropic peptides in crustaceans is still relatively small [for review see (9)]. They include five members of the family of FMRFamide-related peptides (FaRPs) (11,14,29), proctolin (20,22,27), crustacean cardioactive peptide (CCAP) (23,26), and orcokinin (24). Orcokinin was identified in our laboratory as a potent hindgut-activating substance in extracts of the ventral nerve cord of the crayfish, Orconectes limosus, and we have recently reported on its isolation and structural elucidation (24). The primary structure of this peptide, N F D E I D R S G F G F N , has no similarity to that of any other known neuropeptide. A peculiarity is the lack of blocking groups both N- and C-terminally. By use of synthetic orcokinin, the dose dependence of the hindgut-contracting activity was tested. The activating effect on the force of

Requests for reprints should be addressed to Dieter Bungart.

393

394

BUNGART, DIRCKSEN AND KELLER METHOD

Peptide Synthesis Solid-phase synthesis of orcokinin was performed on an automated synthesizer (Applied Biosystems 430 A) with N-t-butoxycarbonyl (Boc)-amino acids (Bachem). The solid phase was a conventional Merrifield resin (Fluka). For details of the synthesis procedure see (24). After deprotection and cleavage of the peptide from the resin, it was purified by HPLC (semipreparative uBondapak C 18, Millipore-Waters). Analogues of orcokinin were synthesized by fluorenyl-methoxy-carbonyl (Fmoc) solid-phase strategy on a manual peptide synthesizer (LKB Biolynx 4175), connected to a personal computer and a spectrophotometer. The Fmoc-amino acid derivatives and the polydimethylacrylamide resin (Novasyn PA) were purchased from Novabiochem (Bad Soden, Germany). Synthesis was carried out according to ( 1). Following synthesis, the peptideresin was thoroughly washed and dried in vacuo over potassium hydroxide. Afterwards the peptide was deprotected and cleaved from the resin in a solution consisting of 95% TFA and 5% thioanisol (6 h at RT). The resin was removed by filtration on a sintered glass funnel under reduced pressure and subsequently the filtrate was evaporated to dryness. It was redissolved in 10% acetic acid and then extracted several times with an equal volume of dried diethyl ether. After lyophilization, the crude peptide was purified by HPLC (semipreparative #Bondapak C I8, Waters). For control of amino acid composition and quantification, a conventional amino acid analyzer (Biotronic LC 5000) operating with orthophthaldialdehyde postcolumn derivatization was used (3). Peptide samples were hydrolyzed at 150°C for 1 h with 6 N HCI (constant boiling, Sigma) in vacuo.

Production q/ Antiserum The antigen was produced by glutaraldehyde coupling of synthetic orcokinin to bovine thyroglobulin. Peptide (991 nmol) and 8.59 mg thyroglobulin (Sigma) were dissolved in 1 ml sterile 0.1 M sodium phosphate buffer (pH 7.4) at 0°C. Under continuous stirring, 50 #1 of a freshly prepared 4% glutaraldehyde solution was slowly added, and the coupling reaction was allowed to proceed overnight at 0°C. To complete the coupling, 8 ul of a 25% glutaraldehyde solution was added to the mixture at room temperature (RT). After 15 min of continuous stirring, the conjugate was dialyzed against several changes of 0.01 M phosphatebuffered saline (10 mM sodium phosphate buffer, pH 7.4, 0.9% NaC1) for 48 h at 4°C. Approximately 125 nmol of coupled peptide was dissolved in 0.75 ml 0.1 M sodium phosphate buffer and emulsified with an equal volume of Freund's complete adjuvant (Sigma). One New Zealand white rabbit was injected sub- and intracutaneously with this emulsion. Four booster injections with smaller amounts of peptide (75-100 nmol) were given during 7 months. The animal was terminally bled under anesthesia 56 days after the last booster injection.

Determination Q[lAntibod), Tiler Small volumes of blood were taken from the ear vein to check the antibody titer development in the serum. For this purpose an indirect, noncompetitive ELISA method was used. In the first step synthetic orcokinin, dissolved in phosphatebuffered saline (PBS: 10 mM Na2HPO4.2H20, 1.7 mM KH2PO4, 136 mM NaCI, 2.7 mM KCI, pH 7.4) was adsorbed to microtiter plate wells (Nunc, Maxisorp FI6) (100 ~1 each well, overnight at 4°C). After washing the plate with PBS, 400 ~,1 of a blocking buffer [2% bovine serum albumin (Fraction V,

Sigma), 0.02% NaN3 in PBS] was added for 8 h. Then the wells were filled with dilutions of antiserum in PBS containing 2~ BSA (R1A grade, Sigma) and 0.02% NaN3 (ELISA sample buffer) (overnight, 4°C). Antiserum was removed by washing the plate five times with 400 ul PBS-T (PBS, 1% Tween 20). A goat-antirabbit peroxidase conjugate (Sigma), dissolved 1:1000 in PBST, was then added and kept in the wells for 3-4 h at RT. After washing with PBS-T, the substrate [4 mg 2,2'amino-bis(3-ethylbenzthiazoline-6-sulfonicacid) (ABTS, Sigma), 2 ~1 30% H202 in 10 ml 0.1 M sodium citrate buffer, pH 4] was added, and readings were taken at 405 nm (Titertek Multiscan MK2).

A[linity Puri/ication and Biotinylation ~?/Antibodies The separation of the IgG fraction from whole immune serum was achieved by use of protein-A-sepharose (Sigma) according to standard procedures (28). The IgG fraction was eluted with 0.85% NaC1 (w/v)/0.58% acetic acid (v/v) and collected in sufficient 1 M phosphate buffer (pH 8) to achieve immediate neutralization. The final lgG concentration was determined spectrophotometrically at 280 nm (12,18). Prior to the biotinylation, 2 mg of antibodies was transferred from phosphate buffer to 0.1 M sodium borate buffer (pH 8.8) by means of Centricon-30 tubes (Amicon). Biotinylation reagent (30 mg) (biotinyl+aminocaproic acid N-hydroxysuccinimide ester, Sigma) was dissolved in I ml dimetfiyl sulfoxide, and from this solution 18 ul were added to the antibody solution (volume: 2 ml). The mixture was agitated for 4 h at RT and then a buffer exchange to 0.1 M phosphate buffer (pH 8) was performed.

Sandwich ELIS.I The noncompetitive, indirect sandwich enzyme immunoassay was pertbrmed as follows. The first antibody solution (100 ul) at a concentration of 20 ~g/ml in 0.1 M sodium phosphate buffer (pH 8) was filled into microtiter plate wells (Nunc, Maxisorp F16) (overnight, 4°C). After washing the plate five times with phosphate buffer, blocking buffer was added (400 ~1 each well, overnight at 4°C). The blocking buffer was removed and 100 ul of samples, standards, and blank, dissolved in ELISA buffer, was added and incubated overnight at 4°C. After washing five times with PBS-T, the second, biotinylated antibody in a concentration of 5 ug/ml in EL1SA buffer was added and the plate was incubated for 5-6 h at 37°C. Again the plate was washed with PBS-T and then a streptavidin peroxidase conjugate (Boehringer), diluted 1:25,000 in PBS-T, was added (1 h, RT). After a final washing step, the H202/ABTS reaction (see above) was allowed to proceed for about 60 min and the absorption was measured at 405 nm.

Tissue Evtraclion Male crayfish, Orconectes lhnosus and Astacus astacus, with a carapace length of about 4 cm and 3.0-3.5 cm, respectively, were anesthetized in ice and dissected under cold crayfish saline (7). Tissue specimens were homogenized in 400 ul of extraction medium (Bennett medium) consisting of 1% formic acid, 5% trifluoroacetic acid (TFA), 1% NaCI in 1 N hydrochloric acid (2). After repeated sonication (Branson sonifier with microtip) the samples were centrifuged in an Eppendorf microfuge (10 rain, 4°C, 12,000 rpm). The supernatants were stored frozen until further use and the pellets were dissolved in 1 N NaOH for the determination of protein contents (21). For ELISA measurements, an aliquot of each supernatant was dried in a speedvac concentrator (Savant, Bachofer) and redissolved in ELISA buffer. Female lobsters, ttomarus americanus, weighing 0.5 kg, were anesthetized in ice and dissected under cold saline (16). The

ORCOKININ IN CRUSTACEAN NERVOUS SYSTEM ~.5

• 1 pmol OK 0 1 0 0 f m o l OK

395

of the chromatographic conditions are given in the legend to Fig. 5. RESULTS

~ o

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0.0

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i

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Dilution of Antiserum FIG. 1. Determination of antibody titer by EEISA. Synthetic orcokinin (OK) (1 pmol and 100 fmol) was coupled to microtiter plate wells and the test antiserum was applied in different dilutions.

tissues were treated in the same way as those of the two crayfish species, except that they were homogenized in a larger volume of extraction medium (4 ml), and the centrifugation was performed in a Beckman centrifuge (model JA-21 C, JA 21 rotor) at 15200 rpm for 30 min. Because of the low titer of orcokinin-like material in the hemolymph, at least 1.5 ml had to be collected to obtain measurable amounts. Hemolymph samples were mixed with two volumes of Bennett medium and centrifuged in a Beckman centrifuge (30 rain, 12,000 rpm). To remove salts and other substances possibly interfering in the assay, the supernatants were applied to a SepPak C 18 cartridge (Millipore-Waters), equilibrated with 0.1 I% TFA. The peptide fraction was eluted with 60% acetonitrile in 0.10% TFA. The eluate was dried and resuspended in ELISA buffer. Recovery of orcokinin was tested by adding standards of synthetic orcokinin to Bennett medium or to blood samples treated in the same way as described above. In both cases, no measurable loss oforcokinin during the passage through the SepPak cartridge was detected.

The antibody titer of the rabbit antiserum was determined by a simple ELISA method. Figure 1 shows that at least 100 fmol oforcokinin could be detected at an antiserum dilution of 1:30,000. Further studies with affinity-purified antibodies confirmed that the lower limit of detection of this type of ELISA is about 80-100 fmol orcokinin (not shown). With the sandwich ELISA method we were able to improve the sensitivity about one hundredfold, i.e., to a lower limit of detection to 1 fmol of orcokinin. The standard curve of the sandwich ELISA is shown in Fig. 2. Depending on the tissue, it was possible to measure the orcokinin content of a 1/100 aliquot of one abdominal ganglion or a 1/5000 aliquot of one hindgut. In the lobster, aliquots 10 times smaller were sufficient. To test whether cross-reacting substances other than orcokinin could have contributed to the orcokinin immunoreactivity (IR) in the tissue extracts, we assayed all the fractions after HPLC separation of an extract from 70 ventral nerve cords of Orconectes limosus (Fig. 3). Orcokinin-IR was detected in five adjacent fractions (20-24), with the main fraction coinciding with the retention time of authentic orcokinin. The immunopositive fractions were pooled and rechromatographed (Fig. 4). After this separation, 95% of the orcokinin-lR was located in three adjacent fractions, which corresponded to the retention time of authentic orcokinin. Separate from this cluster there were a few fractions with low immunoreactivity, which, moreover, was detectable only at much higher concentrations of tissue equivalents. Whether this was caused by compounds sharing similar epitopes with orcokinin is under investigation. To test whether there was immunoreactivity associated with larger proteins that were extracted under nondenaturating conditions, we separated an extract of 25 ventral nerve cords on a Superose-12 gel filtration column by means of FPLC (Fig. 5). The collected fractions were assayed by ELISA. Orcokinin-lR was only detectable in fractions 17-25, which corresponded to the peptide fractions. In the preceding fractions, corresponding to the proteins, no immunoreactivity was measured at all.

High PerJbrmance Liquid Chromatography The chromatographic system consisted of two type 510 pumps, a model 680 solvent programmer, a U6K injector, and a model 481 LC spectrophotometer (all Waters Assoc.). Chromatographic conditions and other details are given in the legends to the chromatograms.

Gel Filtration A gel filtration of an extract from 25 abdominal nerve cords of Orconectes limosus was performed on a Superose- 12 column (Pharmacia). Tile abdominal ganglia were homogenized under repeated sonication in 0.1 M Tris-HC1 buffer (pH 7.6) containing 1 mM EDTA and 0.1 m M DTT and then centrifuged in a Beckman centrifuge (20,000 rpm, 45 rain at 2°C). Prior to the gel filtration the supernatant was concentrated to a volume of about 500 ul (Savant speedvac concentrator). Gel filtration was carried out on a FPLC system (Pharmacia) consisting of a liquid chromatography controller (LCC-500), two pumps (P 500), a control unit (single path monitor UV-1), two optical units (single path monitor UV-I/214), and a fraction collector (Frac-100). Details

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70

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FIG. 2. S t a n d a r d c u r v e o f o r c o k i n i n s a n d w i c h ELISA. M e a n s o f triplicates +_ISD.

396

BUNGART, DIRCKSEN AND KELLER

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Retention Time [min] FIG. 3. Reversed-phase HPLC of an extract of abdominal nerve cords (here aliquot corresponding to seven equivalents) from Orconectes limosus and localization of orcokinin-IR. The SepPak-purified material was applied to a #Bondapak phenyl column (Waters), which was equilibrated with 90% solvent A (0.11% TFA in H20) and 10% solvent B (60% acetonitrile in 0.1% TFA). The peptides were eluted with a linear gradient from 10-80% solvent B in 60 min. The flow rate was 0.9 ml/min. The eluate was automatically collected in 1.8-ml fractions. Five fractions (21-24) showed orcokinin-lR, the main fraction (21) coinciding with the retention time of orcokinin (arrow). The immunopositive fractions were pooled and rechromatographed (Fig. 4). AUFS (absorption units full scale).

The specificity of the polyclonal antiserum was further tested by use of N- and C-terminally deleted analogues of orcokinin. The cross-reactivity of the analogues in the standard sandwich ELISA is shown in Figs. 6 and 7. The antiserum showed a high specificity to orcokinin, because even these closely related analogues were not recognized to a comparable degree, Furthermore, it is clearly shown that the antibodies are directed against the C-terminal region of the peptide because the deletion of one C-terminal amino acid (OK C- 1) led to a sharp decrease of binding (Fig. 7). Removal of a further amino acid (OK C-2) resulted in very low and essentially concentration-independent absorptions. In contrast, N-terminal deletions of one and two amino acids (OK N-l, OK N-2) did not decrease binding as dramatically. The analogues OK N-3 and OK N-4 were, however, hardly recognized by the antibodies.

Distribution of Orcokinin-IR in the Nervous System Orconectes limosus. The central nervous system of astacidean crustaceans consists of 15 ganglia joined by connectives. The brain is the first and largest of these ganglia, and is followed by two connective ganglia, the subesophageal ganglion (SEG), five thoracic ganglia (TG 1-5), and six abdominal ganglia (AG 16). Segmental nerve roots arise in a regular pattern from the TG and the first five AG. They connect the ventral nerve cord with peripheral structures. The stomatogastric nervous system consists of two small ganglia, the stomatogastric (STG) and esophageal (EG) ganglia, within a network that is connected to the connective ganglia within the brain-SEG connectives (CON) and the brain. The innervation of the esophagus and stomach arises from

the EG and STG, respectively• The eyestalk ganglia (ESG) were dissected as a whole, including the sinus gland. Orcokinin-IR was detectable throughout the entire nervous system (Table 1). The highest amount was measured in the hindgut, followed by the brain, the SEG, and the eyestalks. From the brain and SEG to the thoracic and abdominal ganglia of the ventral nerve cord, a stepwise decrease of orcokinin-IR was observed• The AG 1-5 contained relatively low amounts of orcokinin-lR but, remarkably, the content of the sixth AG was about five times higher than that in each of the other AG, When the amount of immunoreactivity was normalized to the protein content, a similar pattern of distribution was apparent. A marked decrease was only visible in the hindgut, because of the high tissue mass of this organ compared to that of the ganglia of the nervous system• The orcokinin-lR measured by ELISA was specific to the nervous system, because in other tissues-heart, abdominal muscle, and hepatopancreas--no immunoreactivity was detectable. For the determination of orcokinin-lR in the hemolymph at least 1-2 ml of hemolymph had to be collected. After SepPak purification, an amount of approximately 10 fmol/ml hemolymph was determined• This corresponds to a concentration of approximately 1 × 10T M M. Astacus astacus. This species gave results similar to those in Orconectes limosus, which was not surprising in view of the close relationship• Orcokinin-IR was also detectable in the entire nervous system, and the general distribution, beginning with high concentrations in the SEG, brain, and eyestalk ganglia to the lowest concentrations in the AG 1-5, is nearly identical (Table 1). Measurements in hindgut extracts also gave the highest absolute amounts of orcokinin-IR, The animals were smaller

ORCOKININ IN CRUSTACEAN NERVOUS SYSTEM

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FIG. 4. Rechromatography of an extract of pooled immunoreactive fractions from five HPLC runs as shown in Fig. 3 on a Baker C18 wide pore column (250 × 4.6 mm), which was equilibrated with 65% solvent A and 35% solvent B. Elution was carried out with a linear gradient from 35-70% solvent B in 60 min. The flow rate was 0.9 ml/min. The eluatc was fractionated by hand. Approximately 95% of the whole immunoreactivity was located in three adjacent fractions, coinciding with the retention time of orcokinin (arrow).

than Orconectes fimosus, which resulted in somewhat lower absolute amounts oforcokinin-IR in the ganglia. Values normalized to protein (pmol/mg protein) clearly showed that the content of orcokinin-IR in the different ganglia is almost identical to that

0.5

of Orconectes limosus. Nevertheless, it was striking that the brain and the AG 1-5 contained about three to four times lower concentrations. On the other hand, the enrichment of orcokininIR in the AG 6 was 20-fold over the other AG and thus more pronounced than in Orconectes limosus. Homarus americanus As a further representative of the astacidean decapods, the lobster, Homarus americanus, was investigated. Orcokinin-IR was also detectable in every portion of

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Fraction FIG. 5. Gel filtrationon a Supcrosc- 12 column (Phannacia) of an extract of 25 abdominal nerve cords from Orconectes limosus by F P L C and localization of orcokinin-IR. P B S buffer (pH 7.6) was used as mobile phase at a flow rate of 0.2 ml/min. The eluate was collected in 0.5-ml fractions. Orcokinin-lR was detectable in the peptidc fractions but not in the protein fractions.

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was found in the SEG, followed by the eyestalks, the hindguL a n d the brain. In agreement with the crayfish species, the lowest c o n t e n t of orcokinin-IR was measured in the AG. The i m m u noreactivity measured in the sixth A G was not significantly higher t h a n that of the preceding AG. T h e lobster offered the possibility to analyze the pericardial organs (PO), which were difficult to dissect in Orconectes a n d Astacus. They are located in the lateral pericardial cavity and consist of anastomoses of nerve fibers originating from segmental nerves of the TG. T h e i m m u n o r e a c t i v i t y measured in the POs was relatively low a n d in the range of that of the AG, U p o n normalization to protein content, the picture of the distribution o f o r c o k i n i n changed considerably only with regard to the hindgut a n d the eyestalks, which showed low concentrations. O n the other hand, it b e c a m e obvious that the POs were enriched in orcokinin c o m p a r e d to larger tissues like the brain a n d the SEG. In the a b d o m i n a l nerve cord, the sixth A G is not diffi~rent from the other A G in terms o f o r c o k i n i n concentration. In general, the normalized values were in the same range as those d e t e r m i n e d for Orconectes limosus. For m e a s u r e m e n t of orcokinin-lR in the h e m o l y m p h , samples from three animals were collected and processed as described above. Triplicate d e t e r m i n a t i o n s were performed with SepPakpurified material from 2 ml of h e m o l y m p h from each animal. A m o u n t s of 1.43, 1.65, and 1.72 × 10 ~L~ M, respectively, were found, which corresponds to a m e a n of 1.6 × 10 t t M.

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the nervous system. C o m p a r e d to Orconectes limosus, the absolute a m o u n t s of orcokinin-IR in the different ganglia of the nervous system were a b o u t 5 - 1 0 times higher, not surprising in view of the difference in sizes (Table 1). T h e general distribution in the nervous system is nearly the same. T h e largest a m o u n t

TABLE 1 ORCOKININ TISSUE CONTENTS Orconecte.s limosus (n = 5-7)

Astacus o.Macfl.g (n 3)

Tissue

Picomoles

Picomoles per mg Protein

2 ESG Brain CON STG EG SEG POs TG I 2 3 4 5 AG 1 2 3 4 5 6 Hindgut Hemolymph Heart Muscle Hepatop.

16.26 + 1.02 36.02±2.21 3.49 _+ 0.22 0.02 ± 0.00 0.02 ± 0.00 27.05 ± 2.80

38.01 ± 3.77 215.69± 15.46 40.62 _+ 2.50 2.32 ± 0.29 1.83 ± 0.31 182.35 ± 23.29

8.45± 1.14 8.01 ± 0.94 6.13 ± 0.66 3.06±0.15 2.53_+0.33

Picomoles

Picomoles per mg Protein

7.92 ± 3.30 8.78±2.08 1.98 _+ 0.33

38.67 _+ 20.90 71.83± 15.18 36.87 ± 1.00

12.03 ± 4.10

116,89 ± 39.69

65.53± 72.08 _+ 60.38 _+ 35.01± 44.74±

3,81 6.87 4.50 2.72 6.08

4.46±0.87 3.89 _+ 0.44 3.03 ± 0.71 1.13_+0.50 0.36±0.17

48.72±24.12 51.52 ± 11.40 33.57 ± 5.80 17.16_+ 7.51 4.87± 2.54

0.88 +0.04 17.03+_ 1.13_+0.13 15.91_+ 0.76_+0.03 11.07± 0.70_+0.04 13.68± 0.72 ± 0.10 14.06 ± 4.48 +_ 0.53 56.86 _+ 55.04±5.60 22.73_+ I × 10 ~l M . . . . ---

1.91 2.13 0.41 1.32 1.55 6.90 2.81

0.23+0.05 0.14+0.03 0.11_+0.07 0.19_+0.06 0.08 ± 0.04 2.99 _+ 0.61 33.62±2.28

2.65± 0.14 3.52_+ 0.29 3.63± 1.60 4.37_+ 0.75 2.46 _+ 1.16 71.78 _+ 19.50 32.95± 6.80

. .

. .

. .

Homurus

ami,ri(

attl4s

(n - 4) Picomoles 202.87 + 23.49 89.02+ 7.77 13.56 _+ 0.79 0.60 ± 0.09 223.44 _+ 27.35 8.61 + 4.40 56.90± 33.20 ± 27.81 ± 28.55 + 25.66±

11.16 2.42 4.75 3.13 1.13

15.35 + 2.21 16.09 +_ 1.25 12.26+ 1.84 10.21 + 1.61 7.66 ± 1.00 17.88 _+ 2.04 182.67± 11.98 1.6 x 10-t]

Picomoles per mg Protein 39.65 ± 81.21_+ 25.06 _+ 2.28 2

6.33 3.59 3.31 0.20

221.58 ± 43.24 61.27 ± 40.82 59.56_+ 18.77 39.10 _+ 2.73 39.09 ± 9.96 41.37+_ 8.20 39.45_+ 4.50 33.73 + 25.22_+ 23.15_+ 18.87± 13.66 + 25.16 _+ 11.81 _+ M

5.88 1.91 3.69 1.57 1,87 4,88 1.83

. .

Values are mean _+ SEM. ESG (eyestalk ganglia), CON (connectives brain to SEG), STG (stomatogastric ganglion), EG (esophageal ganglion), SEG (subesophageal ganglion), PO (pericardial organ), TG (thoracic ganglion), AG (abdominal ganglion).

ORCOKININ IN CRUSTACEAN NERVOUS SYSTEM

399

To check if the immunoreactive material in the lobster is really orcokinin, we separated an extract of one brain by HPLC and determined the immunoreactivity in all the fractions by ELISA (Fig. 8). The main reactive fraction (number 36) coincided with the retention time of authentic orcokinin.

both findings clearly support a neuromodulator/neurotransmitter role oforcokinin in the regulation of hindgut activity. In the lobster, however, the amount of orcokinin-IR in the sixth AG is not significantly higher than in the preceding AG, although a high level was detected in the hindgut. That the physiological significance of orcokinin is not restricted to this specialized role is indicated by its presence throughout the entire nervous system. Preliminary immunocytochemical mapping studies have identified several types of orcokinin-positive neurons, which clearly suggests a central nervous role of this peptide (5). Comparable investigations on the quantitative distribution of FLI, a peptide of the FaRP family ( 10,13), proctolin (20), and crustacean cardioactive peptide (CCAP) in the nervous system of Orconectes limosus (26) and the lobster (unpublished results from our laboratory) are available. The level of orcokinin is generally higher than that of the other peptides, except for the pericardial organs (POs) (see below). Mercier et al. (13) found levels of FLI in the nervous system of the crayfish, Procambarus clarkii, about 10-fold higher than Kobierski et al. (10)detected in the lobster. Thus, FLI concentrations in Procambarus are closer to those found in the present study for orcokinin, a major difference being the very high content of FLI in the POs. In the AG, the levels of CCAP are in the same range as those of orcokinin, whereas in the brain, the SEG, and the TG it is much lower. Orcokinin resembles CCAP in being present in the sixth AG in particularly high concentrations (26). In contrast, the levels of FLI and proctolin are not particularly high in the AG 6. This suggests that both orcokinin and CCAP have a specific role in the regulation of hindgut contractions. As in the case of other crustacean neuropeptides, a role of orcokinin as a circulating neurohormone must also be considered. In this respect, it is of interest that immunoreactive peptide could reliably and reproducibly be detected in the hemolymph, at levels of approximately 1 × 10 1~ M, close to the threshold concentration for excitation of the crayfish hindgut (24). This

DISCUSSION

The development of a sensitive and specific ELISA enabled us to determine the distribution oforcokinin, a recently discovered myotropic neuropeptide (24), in the different parts of the nervous system of two crayfish species and in another important astacidean crustacean, the lobster, Homarus americanus. The immunoreactive material in the lobster seems to be authentic orcokinin, because immunoreactivity was found to be almost completely associated with a fraction having the same retention time as the synthetic peptide on HPLC. In all species, orcokinin-IR was detectable throughout the entire nervous system. As expected in view of the size differences, the absolute amounts of orcokinin-IR in the lobster nervous system are about 10 times higher than those in the crayfish species, but its distribution is nearly the same. When the absolute amounts were related to the size of the tissues by normalization to protein content, it became apparent that the values, expressed per mg of protein, were in the same range in all three species. In terms of distribution, the eyestalks, the brain, and the SEG have higher levels than the TG, and these, in turn, have somewhat higher levels than the AG 1-5. In Orconectes limosus and Astacus astacus, the sixth AG, from which innervating nerve fibers to the hindgut arise (6), contains about five times (Orconectes) and 20 times (Astacus) higher levels of orcokinin-IR than each of the preceding AG. In immunocytochemical investigations in our laboratory, the abundant innervation of the hindgut with orcokinin-immunoreactive fibers from the AG 6 have been demonstrated (5). This result agrees well with the high contents oforcokinin-IR in the hindgut, as determined in this study, and

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Retention T i m e [min] FIG. 8. R P - H P L C of an extractof one brain from the lobster,Homarus americanus, and localizationof orcokinin-IR. The ScpPak-pufified material was applied to a Baker C 18 wide pore column, which was equilibratedwith 90% solvcnt A and 10% solvent B. Elution was carded out with a lineargradient from I0-80% solvent B in 60 rain. The flow ratewas 0.9 ml/min. Thc eluatewas automaticallycollectedin l-ml fractions. The main immunoreactive fraction corresponds to the retention time of authentic orcokinin.

400

BUNGART, DIRCKSEN AND KELLER

concentration is similar to those found for other neuropeptides, i.e., proctolin (20), FMRFamide-like peptides [FLI (10)] in the lobster hemolymph, and C C A P in the crayfish and shore crab hemolymph (25,26). What is the principal source of these circulating peptides? The fact that the POs of the respective species have by far the highest levels of proctolin, FLI, and CCAP (10,20,25,26) makes these extended neurohemal structures [for review see (4)] the prime candidates, although there may be contributions from other release sites, e.g., in the case of FLI, the neurosecretory region in the thoracic second roots and in the connective sheath of the ventral nerve cord (10), Concerning orcokinin, the POs from H o m a r u s were analyzed in this study. In striking contrast to the above-mentioned peptides, orcokinin levels were low, which appears to rule out the POs as the principal source of hemolymph orcokinin. Preliminary im-

munocytochemical results in our laboratory suggest other possibilities, e.g., neurohemal areas in the SEG and the T G (5). These results will be published in a separate paper. Obviously, it is of interest to find out whether orcokinin or similar peptides occur among crustaceans other than the three astacidean species or other arthropods and invertebrates. First attempts to detect it in a brachyuran crustacean, ('arcinus m a e n a s , have met with failure, but there is some preliminary evidence of the presence of an analogue of orcokinin in this species, which does not readily cross-react in the ELISA (in preparation). ACKNOWLEDGEMENT This work was supported by a grant from the Deutsche Forschungsgemeinschaft (Ke 206/7-9) to R.K.

REFERENCES 1. Atherton, E.; Sheppard, R. C. Solide phase peptide synthesis: A practical approach. Oxford: IRL Press; 1989. 2. Bennett, H. P. J. Peptide extraction methods. In: Gross, B.; Brown, C. A.; Goltzmann, D.; Solomon, S., eds. Peptides: Structure and biological functions. Rockford, IL: Pierce; 1979:121-131. 3. Benson, J. R.; Louie, R.; Bradshaw, R. A. Amino acid analysis of peptides. In: Gross, E.; Meienhofer, J., eds. The peptides. Analysis, synthesis, biology, vol. 4. New York: Academic Press; 1981:217260. 4. Cooke, I. M.; Sullivan, R. E. Hormones and neurosecretion. In: Atwood, H.; Sandeman, D., eds. The biology of crustacea, vol. 3. New York: Academic Press; 1982:205-391. 5. Dircksen, H.; Bungart, D.; Stangier, J.; Keller, R. Immunocytochemistry, quantification and biological functions of orcokinin, a novel crustacean myotropic peptide, in the crayfish Orconectes limosus. In: Elsner, N.; Richter, D. W., eds. Proceedings of the 20th G0ttingen Neurobiology Conference. Stuttgart: Thieme; 1992:515. 6. Elekes, K.; Florey, E.; Cahill, M. A. Morphology and central synaptic connections of the efferent neurons innervating the crayfish hindgut. Cell Tissue Res. 254:369-379; 1988. 7. Harreveld van, A. A physiological solution for freshwater crustaceans. Proc. Exp. Biol. 34:428-432; 1936. 8. Holman, G. M.; Nachman, R. J.; Wright, M.; Schoofs, L.; Hayes, T. K.; De Loof, A. Insect myotropic peptides: Isolation, structural characterization and biological activities. In: Menn, J. J.; Kelly, T. J.; Masler, E, P., eds. Insect neuropeptides: Chemistry, biology and action. Washington, DC: American Chemical Society; 1991: 40-50. 9. Keller, R. Crustacean neuropeptides: Structures, functions and comparative aspects. Experientia 48:439-448; 1992. 10. Kobierski, L. A.; Beltz, B. S.: Trimmer, B. A.; Kravitz, E. A. FMRFamidelike peptides of Homarus americanus: Distribution, immunocytochemical mapping, and ultrastructural localization in terminal varicosities. J. Comp. Neurol. 266: l- 15; 1987. 11. Krajniak, K. G. The identification and structure-activity relations of a cardioactive FMRFamide related peptide from the blue crab Callinectes sapidus. Peptides 12:1295-1302; 1991. 12. Layne, E. Spectrometric and turbidimetric methods of measuring proteins. Methods Enzymol. 3:447-454: 1957. 13. Mercier, A. J.; Orchard, l.; TeBrugge, V. FMRFamide-like immunoreactivity in the crayfish nervous system. J. Exp. Biol. 156:519538; 1991. 14. Mercier, A. J.; Orchard, l.; Te Brugge, V.; Skerrett, M. Isolation of two FMRFamide-related peptides from crayfish pericardial organs. Peptides 14:i31-135; 1993.

15. Muneoka, Y.; Kobayashi, M. Comparative aspects of structure and action of molluscan neuropeptides. Experientia 48:448-456; 1992. 16. Nordmann, J. J.; Morris, J. F. Depletion ofneurosecretory granules and membrane retrieval in the sinus gland of the crab. Cell Tissue Res. 205:31-42; 1980. 17. Orchard, l.; Belanger, J. H.; Lange, A. B. Proctolin: A review with emphasis on insects. J. Neurobiol. 20:234-254; 1989. 18. Peterson, G. L. Determination of total protein. Methods Enzymol. 91:95-119; 1983. 19. Price, D. A.; Greenberg, M. J. The hunting of the FaRPs: The distribution of FMRFamide-related peptides. Biol. Bull. 177:198-205; 1989. 20. Schwarz, T. L.; Lee, G. M.; Siwicki, K,; Standaert, D. G.; Kravitz, E. A. Proctolin in the lobster: The distribution, release and chemical characterization of a likely neurohormone. J. Neurosci. 4:1300-1311; 1984. 2 I. Smith, P. K.; Krohn, R. 1.; Heramanson, G. T.; et al. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150:76-85; 1985. 22. Stangier, J.; Dircksen, H.; Keller, R. Identification and immunocytochemical localization of proctolin in pericardia[ organs of the shore crab, Carcinus maenas. Peptides 7:67-72; 1986. 23. Stangier, J.; Hilbich, C.; Beyreuther, K.: Keller, R. Unusual cardioactive peptide (CCAP) from the pericardial organs of the shore crab, Carcinus maenas. Proc. Natl. Acad. Sci. USA 84:575-579; 1987. 24. Stangier, J.; Hilbich, C.; Burdzik, S.; Keller, R. Orcokinin: A novel myotropic peptide from the nervous system of the crayfish, Orconectes limosus. Peptides 13:859-864; 1992. 25. Stangier, J.; Hilbich, C.; Dircksen, H.; Keller, R. Distribution of a novel cardioactive neuropeptide (CCAP) in the nervous system of the shore crab, Carcinus maenas. Peptides 9:795-800; 1988. 26. Stangier, J.; Keller, R. Occurrence of the crustacean cardioactive peptide (CCAP) in the nervous system of the crayfish, Orconectes limosus. In: Wiese, K.; Krenz, W. D.; Tautz, J.; Reichert, H.; Mulloney, B., eds. Frontiers in crustacean neurobiology. Basel: Birkh~iuser; 1990:394-399. 27. Sullivan, R. E. A proctolin-like peptide in crab pericardial organs. J. Exp. Zool. 210:543-552; 1979. 28. Tijssen, P. Practice and theory of enzyme immunoassays. In: Burdon, R. H.; Knippenberg van, P. H., eds. Laboratory techniques in biochemistry and molecular biology. Amsterdam: Elsevier; 1985:105107. 29. Trimmer, B. A.; Kobierski, C. A.; Kravitz, E. A. Purification and characterization of immunoreactive substances from the lobster nervous system: Isolation and sequence analysis of two closely related peptides. J. Comp. Neurol. 266:16-26; 1987.