Distribution and coexistence of urotensin I and urotensin II peptides in the cerebral ganglia of Aplysia californica

Peptides,Vol. 13, pp. 695-703, 1992 Printedin the USA.

0196-9781/92 $5.00 + .00 Copyright© 1992PergamonPressLtd.

Distribution and Coexistence of Urotensin I and Urotensin II Peptides in the Cerebral Ganglia of Aplysia californica G. C. G O N Z A L E Z , *l M. M A R T I N E Z - P A D R O N , .2 K. LEDERISi" A N D K. L U K O W I A K *

Departments of*Physiology and iPharmacology, Faculty of Medicine, The University of Calgary, 3330 Hospital Drive iV. IV., Calgary, Alberta, Canada T2N 4NI Received 21 F e b r u a r y 1992 GONZALEZ, G. C., M. MARTINEZ-PADRON, K. LEDERIS AND K. LUKOWIAK. Distribution and coexistence ofurotensin 1 and urotensin Hpeptides in the cerebralganglia ofAplysia californica. PEPTIDES 13(4) 695-703, 1992.--Urotensin I (UI) and urotensin II (UII) were demonstrated in the cerebral ganglia of Aplysia californica by applying immunocytochemical and radioimmunoassay procedures. Sequential analysis of adjacent sections of the cerebral gangliaof Aplysia demonstrated that the UIimmunoreactive (UI-IR) neurons of the F cluster oftbe cerebral gangliaalso contained UII immunoreactivity (UII-IR). Both UIIR and UII-IR were also observed in a cuff-likearrangement of fibers surrounding the proximal portion of the supralabial nerve, as well as in a few fibers in the anterior tentacular nerves. The UI-IR perikarya of the cerebral ganglia appeared to project to the entire CNS of Aplysia, but the UII-IR fibers appeared only in the neuropile and commissure of the cerebral ganglia. The UI-IR staining was abolished by previous immunoabsorption of the UI antiserum with sucker (Catastomus commersoni) UI, but not with ovine corticotropin-releasingfactor (CRF), rat/human CRF, or goby (Gillichthys rnirabilis)UII. Immunostaining with UII antiserum was quenched by goby UII, but not by sucker UII-A, UII-B, UlI-A(6-12), or carp (Cyprinus carpio) UlI-a and UII-7. The UII staining was not abolished by UI or somatostatin. The F cluster was not stained when a somatostatin antiserum was applied. Radioimmunoassay of dilutions of cerebral ganglia extract, using UII antiserum, revealed a parallel displacement curve to synthetic goby UII. These results suggest: i) that both UI- and UII-like substances might exist in the cerebral ganglia of Aplysia californica; ii) that putative Aplysia UI and UII peptides are probably sharing epitopes with sucker (C. commersoni)UI and goby (G. mirabilis) UII, respectively;iii) that a UIl-like substance coexists with UI in the same neurons of the F cluster of the cerebral ganglia of Aplysia; and iv) that the UIl-like substance originating in the cells of the F cluster may have an integrated role with a still unknown action of a putative UI in the CNS ofAplysia californica. Urotensin I Urotensin I1 Cerebral ganglia

Immunocytochemistry

Radioimmunoassay

SINCE Scharrer (43) first investigated the role of neurosecretory systems in molluscs, numerous neuropeptides that were first identified in vertebrates have been demonstrated immunologically in several species of molluscs, includingAplysia californica (32,35,41,42,45). Some studies in invertebrates indicated that corticotropin-releasing factor (CRF)-like peptides may exist in the cockroach Periplaneta americana (18,44,48) and in the cricket Gryllus bimaculatus (18). In a previous work we have demonstrated, for the first time, the presence of urotensin I (a CRF-like peptide) in the CNS of Aplysia californica by using radioimmunoassay (RIA) and immunocytochemistry (ICC) (10,11). Urotensins are neuropeptides isolated from urophysis, the neurohemal organ ofa neurosecretory system in the caudal spinal cord of teleost, elasmobranch, and ganoid fishes. Urotensin I

Aplysiacalifornica

(UI) is a 41 amino acid peptide isolated from urophysis of the freshwater fishes Catastomus commersoni (24) and Cyprinus carpio (13), as well as from the marine fishes Hyppoglossoides elassodon (29) and Platichthys flesus (5). All these UIs share more than 50% structural homology with all mammalian CRFs and frog (Phyllomedusa sauvaget) sauvagine (SVG) (9). The three peptides, fish UI, frog SVG, and mammalian CRF, were able to stimulate the in vitro as well as the in vivo release of ACTH when using mammalian pituitary dispersed cells and CRF-suppressed rats, respectively (40). In addition, in vitro and in vivo studies in the gold fish (Carassius auratus) have demonstrated that UI stimulates the release of ACTH from the teleost pituitary (8). Apart from that effect, UI seemed to also have an osmoregulatory function in fishes (25), as well as a unique vasodilatory action in mammals (26).

Requests for reprints should be addressed to G. C. Gonz,5.tez. 2 Present address: Neurobiologie CeUulaireet Moleculaire, CNRS, 91198 Gif-sur-Yvette, France.

695

m~

z

UROTENSIN I/II IN Aplysia CEREBRAL G A N G L I A

Urotensin II (UII) is a 12 amino acid cyclic peptide, also isolated from the fish caudal neurosecretory system. At the present time, nine structural forms of UII have been isolated from goby Gillichthys mirabilis (gUll) (3), carp Cyprinus carpio (cUlla, cUII-31, cUII-B2, and cUll-y) (14), sucker Catastomus commersoni (sUII-A and sUII-B) (27), flounder Platichytes flesus (fUll) (4), and common dogfish Scyliorhinus canicula (dUll) (6). All nine forms have complete homology at the ring structure of the peptide [UII(6-11)] and C-terminus but they differ at the N-terminus (6,14,27). Urotensin II appeared to have smooth muscle-contracting activity in fish, including a hypertensive response (1,23,53), and like UI, UII also has an osmoregulatory function in fishes (25). Recently, a potent contractile effect and functional receptors of UII have been found in rat thoracic aorta, abdominal aorta, and mesenteric artery (17). Expression of functional UII receptors has also been found in the avian amnion (2). The presence of UI as well as UII has been demonstrated by ICC and RIA in the Dahlgren cells of the caudal neurosecretory system of a number of elasmobranchiomorphe species (33,34,36) as well as teleostome fishes (7,20,21,33,34,37,47,50). The genes encoding for UI and UII have also been demonstrated in the carp (C. carpio) caudal neurosecretory system by in situ hybridization (15) or cDNA recombinant technology (30). Furthermore, urotensin I and UII appeared to be colocalized with various degrees of coexistence in the neurons of the caudal neurosecretory system of elasmobranch fishes Raya binoculata (34) and Triabis scyllia (36), as well as teleost fishes Porichthys notatus (34), G. mirabilis (22), C. commersoni (51), and Cyprinus carpio (15). Recently, however, an extraurophyseal distribution of UI in the fish brain (20,28,47,50), and of UII in CSF-contacting neuronal perikarya in the very rostral spinal cord (just caudal to the obex) of C. commersoni (51), has been reported. The present study was carried out to get more detailed information about UI distribution in the cerebral ganglia and to investigate whether or not UII was also present in Aplysia californica CNS, and if present what would be its relation to previously detected UI-like structures in the CNS of Aplysia. METHOD

Immunocytochemistry Adult Aplysia californica (110-260 g) were obtained from Sea Life Supply (Sand City, CA) and kept in a 1200-1 aerated aquarium containing artificial sea water (ASW, Instant Ocean) at 15-17°C. Prior to dissection, the animals were anesthetized by injection of isotonic MgCl2 (33% of body mass). Circumesophageal ganglia were dissected out and pinned out on Sylgard-

697 coated dishes containing ASW. After replacement of the ASW, the tissues were fixed by immersion in complete Bouin's fixative followed by postfixation in Bouin's without acetic acid. The cerebral ganglia were separated, under dissection microscope, dehydrated, cleared in xylene, and embedded in paraffin using horizontal, sagittal, and coronal planes. Some paraffin blocks containing the embedded cerebral ganglia were used to cut 7-#m paraffin sections with a 820 Spencer microtome (American Optical Corp.). Other blocks were properly carved on the side opposed to the cutting face, mounted on a cryostat object holder by using Tissue-Tek O.C.T. compound (Miles Inc.), and stored at - 7 0 ° C for 30 min before cutting. A roll of 2-#m sections of the cerebral ganglia was obtained using a wooden cylinder adapted to a 2800 Frigocut E (Reichert-Jung) cryostat. The temperatures in the box and of the object were - 5 and - 10°C, respectively. The roll was extended on a fiat surface and 2-#m adjacent sections were separated using a fine scalpel. Adjacent section methodology [see (12)] was applied to investigate the presence of UI and UII immunoreactivities using 5D~ and 4Y2 antisera, respectively, on those consecutive 2-#m sections. The procedure for obtaining thick (40 #m) sections was as follows: after dissecting out the ganglia, they were washed in Tris-phosphate-buffered saline (TPBS: 8 m M Na2HPO4, 3 m M KH2PO4, 120 m M NaCI, 40 m M Tris-base, adjusted to pH 7.78 with concentrated HCI), immersed in TPBS containing 30% sucrose at 4°C, and finally embedded in Tissue-Tek for 30 min at - 7 0 ° C before cutting in the cryostat set at -20°C. All sections were mounted on gelatin-coated slides and exposed to 10% H202, to reduce endogenous peroxidase activity before applying immunostaining. Immunostaining was performed using the Stenberger's peroxidase- antiperoxidase (PAP) procedure as modified by Sofroniew and Glassman (46). The UI primary antiserum (5D3 was raised in rabbits against nonconjugated sucker (C. commersoni) UI (sUI). Characterization by RIA showed complete cross-reaction with sucker and carp UIs and not detectable crossreactivity with ovine CRF (oCRF) or goby (Gillichtys mirabilis) UII (11). Urotensin II primary antiserum (4Y2) was raised against synthetic goby (G. mirabilis) UII (gUII) conjugated to several carrier proteins (21). These include porcine thyroglobulin, bovine serum albumin (fraction V), ovalbumin (grade VI), all purchased from Sigma (St. Louis, MO), and keyhole limpet hemocyanin from Calbiochem (La Jolla, CA). Characterization by RIA (2 I) showed complete cross-reaction with goby UII, sucker (C. commersoni) UII-A (sUII-A) and UII-B (sUII-B), carp (Cyprinus carpio) UIIa (cUII-a), -3 (cUII-3), and -3, (cUII-~,), and the sUII-A flag-

FIG. 1. Photomicrograph of an oblique section passing through the cerebral ganglia ofAplysia californica showing UII-IR perikarya in the F cluster (F). Note that the UII-IR fibers project into the neuropile of both hemiganglia, x 104. FIG. 2. Longitudinal section by the dorsal aspect of the F cluster (F) showing UI-IR neurons. The UI-IR fibers start appearing more ventrally, increasing their density and staining intensity the closer they are to the commissure of the cerebral ganglia (see Fig. 6). On the top of the picture the H cluster can be seen, rostral to the F clusters. X 110. FIG. 3. Composite photomicrograph of two cerebral hemiganglia sectioned dorsal to the F cluster and displaying a bilateral group of UI-IR cells that appeared to be located between the trunks of the anterior tentacular nerve and cerebro-pedal connective. X45. FIG. 4. Small bilateral group of UI-IR cells (arrowheads) appearing close to the inner wall of the proximal end of the cerebro-pedal connective (CPC). More dorsal sections showed that these UI-IR neurons clearly appear to be located between the trunks of the cerebro-pedal and cerebro-pleural (CPIC) connectives, x45. FIG. 5. The UI-IR perikarya in a 2-#m section through the F cluster of the cerebral ganglia. X 170. FIG. 6. Section ventral to the F cluster and passing through the cerebral commissure. In this longitudinal plane, numerous UI-IR fibers can be seen in the cerebral neuropile (N) and commissure (C). X 115. FIG. 7. The UII-IR neurons in a 2-ttm section adjacent to the section shown in Fig. 5, but stained with 4Y2 antiserum. The arrowheads show UIIIR cells that also displayed UI (5Dr) immunoreactivity in Fig. 5. X170.

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FIG: 8. The UII-IR fibers in the neuropile (N) and commissure (C) of the cerebral ganglia ofAplysia califi~rnica. Note that the number and intensity of staining of these fibers is rather less than the UI-IR fibers shown in Fig. 6. X 107. FIG. 9. Oblique section through the supralabial nerve (SLN) and anterior tentacular nerve (ATN) showing UI-IR fibers terminating in the perineurium of the SLN. Note that one UI-IR fiber (arrowheads) can be seen in the ATN, but no stained fibers were seen in the perineural wall of this nerve. X45. FIG. 10. Oblique section through the cerebral trunks of the ATN and SLN showing UI-IR fibers entering the nerves. Note that only in the SLN the fibers appear to terminate in the perineural wall (arrowheads). x 110. FIG. 11. Section adjacent to the section shown in Fig. 9, but stained for UII. A comparison of Figs. 9 and 11 shows that both UI-IR and UII-IR fibers appear to terminate in the same site in the SLN. In addition, note that the UI-IR fiber in the ATN of Fig. 9 also shows UII-IR in the Fig. 11 (arrowhead). ×45.

ments: 5-12, 6-12, 1-11, 5-11, and 6-11 (the ring structure of UII). The 4Y2 antiserum showed no detectable cross-reactivity with sUI, or urophysin B or D from C. commersoni (21). The sections were incubated with the primary antiserum diluted 1/400 (4Y2) or 1/4000 (5D0 in Tris-phosphate-buffer saline (TPBS) containing Triton X-100 and nongelling carrageenan [see (46)], for 18 h, at r o o m temperature. Taking into account the structural homology between UII and somatostatin (SRIF) [see (38)], three dilutions (1/500, 1/1000, and 1/5000) o f a pri-

mary somatostatin antiserum raised in rabbits against SRIFBSA (UCB Bioproducts, Brussels, Belgium) were also applied to sections of Aplysia cerebral ganglia to investigate whether or not the F cluster was S R I F immunoreactive. Then, the sections were incubated with goat anti-rabbit IgG (Sigma, 1/25, for 30 min) and PAP (DAKO, 1/50, for 30 min) diluted in TPBS/ Triton X-100/carrageenan. The sections were finally developed for 15 min, in the dark, with 0.2% diaminobenzidine solution in TPBS containing 0.001% H202.

UROTENSIN I/II IN Aplysia CEREBRAL G A N G L I A

TABLE 1 EFFECT ON UI AND UII STAININGOF LIQUIDPHASE IMMUNOABSORPTIONSOF 5DL AND 4Y2 ANTISERA,WITH DIFFERENT PEPTIDES AND PROTEINS immunoabsorbedWith

5D~

oCRF r/hCRF sUl gull cull- pc cUI1-T sUII-A sUII-B sUII-A(6-12) SRIF Hemocyanin Thyroglobulin Ovalbumin "r-Globulin Bovine serum albumin

+* +* -* +*

4Y2

+f - f +f +f + i" +* +§ + i" + + :~ + + +

(-) Abolition of the staining; (+) no change in the immunostaining. *f~t§ Final concentrations: *10 #M; ~fl O0uM, ~100 ug/ml; §70 #g/ml. Specificity tests were conducted on a set of adjacent sections on which a usual immunoperoxidase procedure was performed except that the primary antisera was submitted to liquid-phase immunoabsorption either with homologous peptides [5D~ antiserum with sUI, oCRF, or rat/human CRF (r/hCRF); 4Y2 antiserum with gUll, sUII-A, sUII-B, cUll-a, cUll-3', and UII-A(612)] or heterologous peptides (5D~ with gUll; 4Y2 with sUI or SRIF). In addition, preimmunoabsorption controls were performed by preincubating the 4Y2 antiserum with all the protein carriers used in the immunization of rabbits. Among all the proteins used as carriers in the production of 4Y2, only hemocyanin is known to occur in molluscs. Therefore, hemocyanin antibodies in the 4Y2 antiserum were eliminated using immunoaffinity solid-phase adsorption, as follows: hemocyanin was dissolved in a solution containing 0.1 M NaHCO3 pH 8.3/0.5 M NaC1/6 M guanidine-HCl/0.01% Tween 80 (coupling buffer) put in a water bath at 60°C with frequent agitation. The solution was cooled down at room temperature and then added to 2 ml CNBr-activated Sepharose-4B (Sigma), previously preequilibrated with coupling buffer without guanidine-HC1 or Tween 80 and contained in a 2-ml BioRAD disposable polypropylene column. Five mg of hemocyanin per ml of Sepharose were used. Protein plus Sepharose was incubated with end-toend agitation for 1 h at room temperature and 16 h at 4°C. Conjugated Sepharose was washed twice with 10 ml of 50 m M Tris, pH 8.0, at room temperature and incubated with 50 m M Tris pH 8.0/1 Methanolamine for 16 h at 4°C to block remaining active groups. The blocking solution was eluted by washing with 10 ml of buffer without ethanolamine followed by a wash with 50 m M Tris pH 8.0/1 M NaC1/6 M guanidine-HC1 to eliminate nonspecifically bound hemocyanin. Finally the hemocyaninSepharose column was washed with 50 m M Tris pH 8.0 and stored in this buffer at 4°C until used. Immunoaffinity chromatography was performed by incubating the conjugated Sepharose with a 1/20 dilution of 4Y2 antiserum in 50 m M Tris pH 8.0/0.5 MNaC1 for 16 h at 4°C.

699

After elution, the purified antiserum was concentrated using CF25 Centriflo Membrane Cones (Amicon Div., W. R. Grace & Co.). This affinity-purified 4Y2 antiserum was diluted 1/400 in TPBS/Triton X-100/carrageenan for use in immunocytochemistry.

Tissue Extraction Cerebral ganglia were dissected out from 28 adult Aplysia, acetone-dried, weighed, finely cut, and homogenized with a Polytron (Brinkman Instruments) for 5 rain at 4°C in 40 m M acetic acid (19 mg acetone-dried tissue/ml). The homogenate was heated in a boiling water bath for 10 rain and centrifuged at 7000 × g for 5 rain; the supernatant was collected and frozen at -70°C. The extracts were concentrated using a Speed Vac Concentrator attached to a Refrigerator Condensation Trap (Savant Instruments, Inc.). Dilutions of these concentrated extracts were made in 40 m M acetic acid for RIA.

Radioimmunoassay (RIA) and Reagents Iodination of synthetic g u l l and radioimmunoassay were performed essentially as described previously (21) using a LKB (Wallac) 1274 RIAGAMA counter. The 4Y2 antiserum was used at a final dilution of 1/125,000, which gave 50% binding for 12SI-gUIIwith a specific activity of 2 mCi/nmol. Synthetic sUI(141) (PURN 10) was purchased from Bachem (Torrance, CA). Synthetic gUll, sucker UIIs (sUII-A and sUII-B), carp UIIs (cUIIa and cUII-'y), oCRF, r/hCRF, and the fragment sUII-A(6-12) were produced using solid-phase synthetic methodology by Dr. J. Rivier (Salk Institute, San Diego, CA). Somatostatin (SRIF14) was a gift from Drs. Q. J. Pittman and O. P. Rorstad. RESULTS

Immunocytochemistry Urotensin II immunoreactivity (UII-IR) was detected in neurons of the F cluster (Fig. l ). Apart from the F cluster cells, we did not find other UII-IR neurons in the cerebral ganglia. The results of UI immunocytochemistry confirmed the presence of strongly positive neurons in the F cluster oftbe cerebral ganglia of Aplysia (Fig. 2). Two other bilateral groups of UI-IR cells were observed in 40-am sections of the cerebral ganglia: one dorsal to the F cluster between the trunks of the anterior tentacular nerve and cerebro-pedal connective (Fig. 3); the other located ventral to the F cluster and between the proximal ends of the cerebro-pedal and cerebro-pleural connectives (Fig. 4). A comparison of 2-#m adjacent sections of the cerebral ganglia immunostained sequentially for UI and UII revealed that UI-immunoreactive (UI-IR) perikarya of the F cluster also displayed UII immunoreactivity (Figs. 5 and 7). The UI-IR processes projecting to the neuropile and commissure of the cerebral ganglia were thick and strongly stained (Fig. 6). On the other hand, the UII-IR fibers showed a lighter appearance and they seemed to project mostly to the cerebral neuropile and commissure (Figs. 1 and 8), supralabial nerves (SLN) and anterior tentacular nerves (ATN). In the SLN, the UI-IR and UII-IR terminals were seen in the inner side of the perineurium of the proximal portion of the nerve in a cuff-like arrangement of more than 5 mm in length from the cerebral origin of the Cl (SLN) nerves (Figs. 9 and I 1). On the other hand, few UI-IR and UIIIR fibers were seen crossing by the ATN (Figs. 9-1 l). Urotensin I immunoreactivity was completely abolished by preabsorption of the primary 5D1 antiserum with sUI, but it was not prevented by oCRF, r/hCRF, or gUII (Table 1). Uro-

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FIG. 12. A 2-um section passing through the F cluster and after applying the UII (4Y:) primary antiserum before it was submitted to affinity purification. Note that lacunae of the connective tissue sheath appeared stained (arrowheads). × 110. FIG. 13. A 2-urn section adjacent to the section shown in Fig. 12 after applying the UII (4Y2) antiserum previously purified using a hemocyaninSepharose column. Observe that the cells of the F cluster remain immunoreactive, but the staining of the lacunae of the connective tissue has been abolished. × 110.

tensin II staining was quenched by preabsorption of 4Y2 antiserum with gUll, but it was not modified by sucker (C. commersoni) UII-A or UII-B, carp (C. carpio) UII-a or UII-% or the ring fragment of sUII molecule, sUII-A(6-12) (Table 1). Neither was the UII staining modified by SRIF or sUI (Table 1). Liquid-phase preabsorption of 4Y2 antiserum with 100 ~g/ ml of the protein carriers used in the production of the antiserum did not abolish the UII staining (Table 1). Liquid-phase preabsorption of 4Y2 antiserum with hemocyanin and solid-phase affinity chromatography using hemocyanin coupled to CNBractivated Sepharose 4B did not prevent primary UII immunoreaction of the neurons of the F cluster, but completely abolished a secondary reaction of the lacunae of the connective tissue sheath (Figs. 12 and 13). We did not find SRIF-immunoreactive neurons in the F cluster of the cerebral ganglia of Aplysia after applying a SRIF antiserum.

Radioimmunoassay The detection limit of the assay was 1.0 10g UII. Figure 14 shows that the extract of the cerebral ganglia of Aplysia yielded a parallel displacement curve to the dilution curve of synthetic gUll. DISCUSSION The pattern of UI and UII-like immunoreactivities found in the cerebral ganglia ofAplysia californica indicates the presence ofimmunoreactive material that shares epitopes with sucker (G. commersonO UI and goby (G. mirabilis) UII, respectively. The

UI-ICC results, which are not quenched by oCRF or r/hCRF, are supported by RIA findings showing parallel superimposable displacement curves to sUI by extract ofAplysia californica cerebral ganglia (11) using 5Dj antiserum, which does not crossreact with oCRF in either ICC or RIA. The present demonstration o f a Ul-like peptide in the F cluster of the cerebral ganglia, a previous report ( 1 I) showing that the peptide is also present in the C and D clusters of the cerebral ganglia as well as in perikarya of the pleural and abdominal ganglia, and the distribution of UI-IR fibers through the entire CNS ofAplysia californica suggest that a molecule related to the CRF family is likely to be present in this mollusc. In addition, the present findings demonstrate that UI is likely to be present in two other bilateral groups of cells in the cerebral ganglia of Aplysia. Recently, we have observed that a UI-like peptide is also broadly distributed in the CNS of the pond snail Lymnaea stagnalis (Gonzfilez, Martinez-Padrrn and Lukowiak, unpublished results). At the present time, we can only speculate as to how structurally close a putative UI peptide in Aplysia or Lymnaea is to the other known CRF peptides found in vertebrates. The present knowledge of the family ofCRF-like peptides demonstrates that they have been highly conserved during vertebrate evolution. For instance, fish UIs, frog sauvagine, and mammalian CRFs share approximately 50% structural homology and an additional 25% can be derived from each other's molecules by a one-base genetic code substitution. A noticeable example is that r/hCRF and fish (C. commersoni) CRF differed only in 2 amino acid residues (31 ). Recent immunological results suggest that the putative CRF-like molecule found in Aplysia possibly shares epitopes with sucker (C. commersoni) UI (11).

UROTENSIN I/II IN Aplysia CEREBRAL GANGLIA

701

4Y2 1/125,000 50 • ~.

4(2

-_

~ gUll Cerebral gangk

~

.~

30 1:3 .c to a~ 20

10

1

10

100

I 1000

log of gUll or mg Dry-Tissue per Assay Tube

FIG. 14. Semilogarithmic plot comparing competitive inhibition of the 4Y2 antibody t25IgUII binding by extract dilutions of the cerebral ganglia ofAplysia californicaand synthetic gUll.

Localization of CRFs in invertebrates has been reported in the brain, corpora cardiaca, and corpora allata of the cockroach (P. americana) (48) and in the cockroach midgut after applying an antiserum raised against oCRF (44). Urotensin I-like immunoreactivity has also been found in the epithelium of the midgut of the cricket Gryllus bimaculatus and the cockroach P. americana using a UI antiserum cross-reacting with oCRF (18). The present results indicate that a UII-like immunoreactive material is also present in the cerebral ganglia of Aplysia and that it colocalizes with UI-IR material in the perikarya of the F cluster. It was noticeable that both UI-IR and UII-IR fibers were seen terminating in a cuff-like arrangement under the sheath surrounding the proximal segment of the supralabial nerve (SLN), suggesting that a release of the peptides might occur in that area of the CNS ofAplysia californica. On the other hand, the presence of UI- and UII-immunoreactive fibers crossing by the anterior tentacular nerve (ATN) suggests that UI- and UIIlike substances produced in the F cluster can regulate the activity of peripheral organs innervated by that nerve in Aplysia. The sparsity of the UII-IR fibers in the cerebral neuropile and commissure and in the SLN compared to the numerous UI-IR fibers seen in the same sites would suggest that only a small proportion of all the UI-IR fibers also carry UII. The presence of UII in the cerebral ganglia of Aplysia californica is corroborated by UII RIA showing that the displacement curve of dilutions of a cerebral extract is parallel to synthetic gUII curve. All known UII molecules have exactly the same ring structure and their differences are at the N-terminal region [see (6,14,27)]. A previous paper showed that 4Y2 antiserum crossreacted equally well with all known natural and synthetic UII molecules, suggesting that the antiserum had mostly ring structure-directed antibodies (21). However, the present RIA results indicating displacement of gUII by cerebral extract of Aplysia, and the blocking of UII immunostaining by synthetic gUII indicate that 4Y2 antiserum contains N-terminal-directed UII antibodies as well; the inability of sUIIs, cUIIs, and the ring fragment of the known UII molecules to block 4Y2 immunostaining

suggests that the staining might be due to epitopic homology with the N-terminus of the gUII molecule, the only region where the latter peptide differs from the other known UII molecules [see (6,14,27)]. The presence of UII neuropeptide has never been detected before in invertebrates, and even though functional receptors for UII appeared to exist in certain amniotes (2,17), to our knowledge, the presence of UII has not even been detected in vertebrates lacking a caudal neurosecretory system. It has been found that somatostatin shared structural homology with a gUII at positions l and 2, and 7 to 9 (38), but whether or not SRIF is phyletically related to UII remains unknown. Recent evidence demonstrated that there is no sequence homology between the precursors of UII and SRIF-14 in the carp Cyprinus carpio (30), which might indicate that UII and SRIF are encoded by separate genes. Furthermore, recent anatomical data suggest that the synthesis of UII and SRIF might occur in separate cells. For instance, the CSF-contacting UII-IR neurons and SRIF-IR cells in the proximal spinal cord ofCatastomus commersoni and Oncorhynchus kisutch (Teleostei) were completely separated, albeit they were in close spatial interrelationship in which SRIF fibers appeared to innervate the UII neurons (52). We were unable to stain sections of Aplysia cerebral ganglia after applying a somatostatin antiserum, and SRIF peptide did not quench the 4Y2 (UII) staining in the F cluster cells, which indicates that the aplysiid UII-like substance in the cerebral ganglia is not related to somatostatin. Various peptides that were first identified in vertebrates (like CCK, gastrin, Met-enk, AVP, AVT, LHRH, VIP, CGRP, etc.) have been detected by immunochemical procedures in several species of molluscs, including Aplysia californica, Lymnaea stagnalis, Bulla gouldiana, and Helix aspersa (32,35,41,42,45). Previous studies have demonstrated that UI and UII are colocalized in the caudal neurosecretory system of elasmobranch and teleost fishes (15,22,34,36,51 ). In molluscs, coexisting immunoreaction for a-MSH, enkephalin, and FMRFamide has been found in the vena cava of Octopus vulgaris (49). Recently,

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G O N Z A L E Z ET AL.

a peptidergic multitransmitter system where egg-laying hormone coexisted with a-bag cell peptide has been demonstrated in bag cell neurons ofAplysia californica (39). Thus the present findings indicating that UI and UII neuropeptides might be present in the CNS of Aplysia are not totally unexpected. The presence of UI/UII-like peptides in neurons of the F cluster are consistent with the Jahan-Parwar and Fredman (19) hypothesis postulating that neurons of the F cluster may play some role in the modulation of neuronal activity throughout the CNS of Aplysia. Thus the UI- and UII-like peptides originating in the F cluster may have an independent and/or inte-

grated modulatory action within the cerebral ganglia or peripherally throughout the supralabial and anterior tentacular nerves

of Aplysia calfornica. ACKNOWLEDGEMENTS This work was supported by a grant from the Medical Research Council of Canada (MRC). K.L. is a Career Investigator of MRC. M.M.-P. is a recipient of a Fellowship from the Canary Islands Government. The excellent technical assistance of D. Ko in the RIA is gratefully acknowledged. We appreciate the generous supply of somatostatin from Q. J. Pittman and O. P. Rorstad. Thanks are due to Mrs. Donna Shaw for preparation of the manuscript.

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