Urotensin I-like immunoreactivity in amacrine cells of the goldfish retina

Neuroscience Letto'.~, 76 ! 1987) 96 I0(! Elsevier Scientific Publishers Ireland l..td

96

NSL 04538

Urotensin I-like immunoreactivity in amacrine cells of the goldfish retina Masahiro Sakanaka l, Denis McMaster 1, Kuldip Chohan 2, Tamotsu Shibasaki 3, William K. Stell 2 and Karl Lederis l 1Department of Pharmacology and Therapeutics, 2Department of Anatomy, Faculty of Medicine, The University of Calgary, Calgary, Alta. (Canada), and 3Department of Medicine, Tokyo Women's Medical College, Tokyo (Japan) (Received 13 August 1986; Revised version received 24 December 1986; Accepted 27 December 19863

Key words." Urotensin I; Goldfish retina; Radioimmunoassay; High-performance liquid chromatography (HPLC); Corticotropin releasing factor (CRF); Immunohistochemistry Urotensin I-like immunoreactivity (UILI), in different localization from that of corticotropin releasing factor-like immunoreactivity (CRFLI), in the goldfish retina has been demonstrated by means of radioimmunoassay, high-performance liquid chromatography (HPLC) and immunohistochemistry. Radioimmunoassay showed 350+40 pg/mg prot. of UILI in goldfish retina extracts. The immunoreactive material present in the retina was also characterized by reversed phase HPLC. Some of the UILI co-eluted with synthetic carp UI, though the HPLC experiments suggested the existence of other UILI substance(s) with less hydrophobicity than synthetic UI. By immunohistochemistry, UILI and CRFLI were seen in different amacrine cells of the goldfish retina. It is suggested that UI may be involved in the fish visual transmission system together with CRF and other neuropeptides.

Urotensin I (UI), isolated from the urophysis of teleost fish (sucker and carp), exhibits potent hypotensive activity (in mammals) and ACTH releasing activity (mammals and fishes), and it shares a close structural homology with mammalian corticotropin releasing factor (CRF) [3, 7, 18]. Although UI-like immunoreactivity (UILI) has been described in fish brain as well as in the spinal cord [2, 21], its occurrence in fish retina has not been determined. In the present study the presence of UILI in the goldfish retina was investigated by means of radioimmunoassay, HPLC and immunohistochemistry. Furthermore, an attempt was made to determine the occurrence and localization of UILI and CRFLI in the retina, since previous immunohistochemical studies have reported the presence of UILI and CRFLI in fish brain [21] and that of CRFLI in the chicken and rat retinas [5, 11]. Eighty-five dark-adapted goldfish (4-5 inches in body length) were decapitated, the

Correspondence." K. Lederis, Department of Pharmacology and Therapeutics, Faculty of Medicine, The University of Calgary, 3330 Hospital Drive N.W., Calgary, Alta., Canada T2N 4NI. 0304-3940/87/$ 03.50 © 1987 Elsevier Scientific Publishers Ireland Ltd.

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retinas rapidly dissected, and dehydrated in acetone. The retinas (290 mg dry wt.) were homogenized in 0.25% acetic acid (4 ml), the homogenate placed in a boiling water bath for 3 min, cooled and centrifuged at 7000 g for 5 min at room temperature. The supernatant was assayed in triplicate for UILI, using previously described methods [15, 16] and an antiserum raised against synthetic non-conjugated sucker UI [211. The remaining supernatant was frozen, dried down using a Savant vacuum centrifuge and reconstituted in 0.1% trifluoroacetic acid (TFA) (240 td). A sample of 200 ld was applied to reversed phase HPLC using a C-18 iLBondapak column and guard cartridge (Waters). Solvent A was 0.1% TFA in water, solvent B was 60:40 acetonitrile water containing 0.09% TFA, and a linear stepped gradient was used (0 5 min, 0 55% B; 5 35 min, 55 80% B; 35 36 min, 80-100% B; flow rate 1 ml/min). One-minute ( 1 ml) fractions were collected in glass tubes containing 50 l*g BSA in 25 itl water, were frozen, dried by vacuum centrifuge and redissolved in 0.25% acetic acid before assaying in triplicate for UILI as described above. A blank run was collected for UI RIA before the retina extract. Standard peptides were run after the retina extract and detected by UV absorption at 214 nm. For immunocytochemistry, 4 dark-adapted goldfish were killed by decapitation, the retinas dissected out, fixed in Zamboni's solution [22] for 30 min at 4 C , and immersed in 0.1 M phosphate buffer containing 30% sucrose overnight. Frozen sections were cut, mounted on gelatin-coated slides and processed for immunostaining according to the unlabeled antibody peroxidase anti-peroxidase (PAP) method [14] and cobalt glucose oxidase~diaminobenzidine procedures [4, 9] with some modifications [10], employing the UI and CRF antisera described earlier (working dilution 1:2000) [1, 8, 15]. Control sections were first incubated with the antisera which had been pretreated with either excess synthetic UI or CRF (30 40 itg/ml), and processed for immunohistochemistry. 1.0-

o

Carp U| Sucker U|

II

r~

0.5-

110

210 RETENTION TIME

310 [rain}

410

510

Fig. 1. Reversed-phase HPLC of UILI in the goldfish retina. Fractions were assayed using the UI radioimmunoassay as described in the text. The broken line indicates the RIA detection limit (extrapolated to ng/Traction). The arrows indicate the elution positions of synthetic carp and sucker UI.

98

Q

tNL

INL

5 IPL

INL

b

Fig. 2 lal Bright-field photomicrograph showing a pear-like UILI celt in the innermost row of the inner nuclear layer ~INL~. Note weakly stained varicose fibers in the first sublayer (1) of the nner plexiform layer (IPL): × 700. (bl Bright-field photomicrograph showing a fusiform CRFLI cell in the innermost row of the INL. × 700. (c) Bright-field photomicrograph showing a semicircular CRFL1 cell in the innermost row of the INL. which sends a process to the fifdl sublayer (5) of the IPL (arrow): x 700.

Radioimmunoassay showed 3 5 0 + 4 0 pg/mg prot. of U I L I in extracts of goldfish retina ( ~ 25 pg/retina). The U I L I was characterized by reversed phase H P L C and a small part of the UILI co-eluted with synthetic carp UI, but another major peak of U I L I eluted in earlier fractions suggestive of the presence of other U I L I materials with less hydrophobicity than synthetic UI (Fig. 1). About 50% of the 4.2 ng of UIL! applied to H P L C was recovered in the two major U I L I peaks. By immunohistochemistry, UILl cells were seen in the innermost row of the inner nuclear layer. They were characterized by pear-like cell bodies with processes in the first sublayer of the inner plexiform layer (Fig. 2a). In contrast, many C R F L I cells present in the innermost row of the inner nuclear layer showed fusiform perikarya with more intensely stained processes in the first sublayer of the inner plexiform layer than those containing UILI (Fig. 2b), suggesting that U I L I and C R F L I are present in different amacrine cells. Occasionally, weakly stained semi-circular C R F L I cells were seen in the inner nuclear layer, their processes projecting to the fifth sublayer of the inner plexiform layer (Fig. 2c). Pretreatment of the antisera with homologous antigens (UI or CRF) resulted in complete disappearance of the immunostaining. Pretreatment of the UI antiserum with synthetic C R F or of the C R F antiserum with synthetic UI did not affect the immunostaining. The present study has shown U I L I in the amacrine cell layer (inner nuclear layer) of the goldfish retina. Moreover, the immunohistochemical results indicate that UILI is localized in cells different from those containing C R F L I . A relatively low sensitivity of our C R F radioimmunoassay did not permit confirmation of the immunocytochemical findings by RIA. The chemical structure of fish C R F is not known. Therefore, the possibility should be taken into consideration that the inability to detect C R F L I by R I A may be due to structural differences between rat C R F - the antigen used to produce the C R F antiserum and the unknown fish CRF, rather than the limits of sensitivity of our

99 R I A p e r se. R e c o g n i t i o n o f a h e t e r o l o g o u s h o m o l o g u e in i m m u n o c y t o c h e m i s t r y does not necessarily i m p l y the recognition o f such a h o m o l o g o u s s u b s t a n c e at a high dilution o f an a n t i s e r u m for use in an R I A . T h e U I a n t i s e r u m used in the present studies is a case in point: at the d i l u t i o n o f a b o u t 1:1000 in i m m u n o c y t o c h e m i s t r y this antiserum recognizes also ovine or rat C R F , w h e r e a s at a dilution o f a b o u t 1:50,000. in R I A , this a n t i s e r u m shows no significant cross-reactivity with C R F [21]. In the present case it m a y be~zoncluded that the u n k n o w n substance in the U I L I cells is m o r e closely related to U I than to C R F , since the staining was o b s e r v e d after p r e a b s o r b i n g the U! a n t i s e r u m with C R F but not with U I . Still, a prerequisite to a meaningful q u a n t i t a t i o n o f fish C R F m a y be the d e t e r m i n a t i o n o f its a m i n o acid sequence. The H P L C R I A e x p e r i m e n t s showed a large peak o f U I L I , eluting much earlier than a u t h e n t i c U I , whereas the i m m u n o h i s t o c h e m i c a l results d e m o n s t r a t e d only one type o f p e a r - s h a p e d U I L I cells, located in the i n n e r m o s t row o f the a m a c r i n e cell layer. Therefore, all U I L I peaks shown by the H P L C e x p e r i m e n t s m a y be a s s u m e d to derive from the same a m a c r i n e cells. If this is true, a single U I L I cell m a y c o n t a i n f r a g m e n t s a n d p r e c u r s o r s o f U I , a n d / o r U I b o u n d to u n n a m e d carrier p r o t e i n s in a d d i t i o n to a u t h e n t i c U I , as is k n o w n to occur in cells that c o n t a i n p r o - o p i o m e l a n o cortin, o x y t o c i n and vasopressin [6, 12, 17, 19]. The functional significance o f U I L I a n d C R F L I in the goldfish retina remains to be d e t e r m i n e d . It c a n n o t be excluded that they have a role in the visual transmission system o f fish together with o t h e r n e u r o h o r m o n e s a n d p e p t i d e s including s o m a t o s t a tin, e n k e p h a l i n s and s u b s t a n c e P [13]. In p a r t i c u l a r , the possibility that U I and C R F like p e p t i d e s are colocalized a n d co-released with s o m a t o s t a t i n , suggested by structural similarities o f the cells described here to s o m a t o s t a t i n - I R cells [20], is to be expected. This w o r k was s u p p o r t e d by grants from M e d i c a l Research Council ( M R C ) (Cana d a ) to K.L. ( C a r e e r I n v e s t i g a t o r o f M R C ) and to W . K . S . , a n d from A l b e r t a Heritage F o u n d a t i o n for M e d i c a l Research ( A H F M R ) to M.S. ( A H F M R Fellow), who is on leave o f absence from the D e p a r t m e n t o f A n a t o m y at O s a k a M e d i c a l College. The a u t h o r s wish to t h a n k Prof. S. M a g a r i for her interest a n d e n c o u r a g e m e n t . W e are grateful to Mr. D. K o a n d Ms. S. C l a r k for their excellent assistance, and to Mrs. M. Devlin for t y p i n g the m a n u s c r i p t . W e also wish to t h a n k the A H F M R for provision o f H P L C e q u i p m e n t .

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