Radioimmunoassay for salmon pancreatic somatostatin-25

GENERAL

AND

COMPARATIVE

ENDOCRINOLOGY

Radioimmunoassay MARK A. SHERIDAN,*

81,

365-312 (1991)

for Salmon

Pancreatic

CARMEN D. EILERTSON,*

*Department of Zoology, North Dakota and fSchoo1 of Fisheries, University

Somatostatin-25’

AND ERIKA M. PLISETSKAYA~

State University, of Washington,

Fargo, Seattle,

North Dakota 58105’; Washington 98195

Accepted March 21, 1990 A specific and sensitive radioimmunoassay (RIA) for the measurement of plasma levels of somatostatin-25 (SS-25) in salmon was developed using antisera raised against coho salmon (Oncorhynchus kisutch) SS-25. SomatoStatin-25 was iodinated by the chloramine-T method and repurified on Sephadex G-25. The RIA was performed using a double antibody (goat anti-rabbit gammaglobulin as second antibody) method under disequilibrium conditions. Plasma from several salmonids (coho, chinook, rainbow trout, brook trout, arctic char, lake trout, and whitefish) as well as plasma from some nonsalmonids (sucker, bluegill) crossreacted with the antisera; serial dilutions of plasma from rainbow trout, brook trout, chinook salmon, and coho salmon were parallel to the SS-25 standard curve. Plasma from catfish showed negligible cross-reactivity. None of the mammalian somatostatins (somatostatin-14, somatostatin-28) U II, or other pancreatic hormones (insulin, glucagon) tested showed significant cross-reactivity with the antibody in the assay system. The lowest detectable level of LB-25 was 5 pg/tube; especially reproducible results were obtained in the range of 0.15-1.20 &ml, which appears to be the normal range of SS-25 circulating in the plasma of salmonids. Intra- and interassay coefficients of variation were 5.7 and 12.6%. respectively. Injection of glucose into chinook salmon resulted in an elevation of plasma SS-25 titers within 30 min and was coincident with hyperglycemia. o 1w1 Academic press, IIIC.

Somatostatin (SS-14), originally isolated from mammalian hypothalamus (Brazeau et al., 1973), appeared to be a 1Camino acid peptide that inhibited growth hormone release as well as the release of other pituitary, pancreatic, and gut hormones (reviewed by King and Millar, 1979). Since 1973, SS-14 as well as N-terminally extended forms of the somatostatin molecule have been isolated from hypothalamic, pancreatic, and gastrointestinal tissue of a variety of vertebrates, including fish-making it apparent that somatostatin exists in two major forms (reviewed by Conlon, 1989). The smaller form of the peptide exists as a 16amino acid molecule, the structure being highly conserved from fish to mammals. In ’ A portion of this work was presented at the XI International Conference on Comparative Endocrinology, Malaga, Spain, May 14-20, 1989.

fact, the peptides isolated from salmon islets (Plisetskaya et al., 1986), catfish islets (Andrews and Dixon, 1981), anglerfish islets (Noe et al., 1979), and rat pancreas (Benoit et al., 1980) are identical. The larger, N-terminally extended form of the peptide is a more variable molecule and has been found to contain 20, 22, 25, 28, or 34 amino acids. In mammals, the N-terminally extended forms of somatostatin (e.g., somatostatin-28) and SS-14 arise from a single prohormone, preprosomatostatin-I (cf. Argos et al., 1983). Teleost fish examined so far express preprosomatostatin-I (by in large processed to SS-14), and in addition a second prohormone, preprosomatostatinII, which is processed to a 22, 25 or 28 amino acid form, depending on species (reviewed by Conlon, 1989; Plisetskaya, 1989a). In salmon, the N-terminally extended form of somatostatin is somatostat-

365 001~6480/91 $1.50 Copyright 8 1991 by Academic Press, Inc. AU rights of reproduction in any form reserved.

366

SHERIDAN,

EILERTSON,

in-25 (&S-25), which contains the sequence [Tyr7 Gly’O] somatostatin-14 at the carboxy terminus (Plisetskaya et al., 1986). The importance of somatostatins as regulators of metabolism, due to their actions on islet secretion and their peripheral effects, is becoming evident. In coho salmon (Oncorhynchus kisutch), for example, sSS25 has been reported to inhibit insulin release, an action associated with enhanced lipid and carbohydrate breakdown and resulting hyperlipidemia and hyperglycemia, respectively (Sheridan et al., 1987). In addition, SS-14 has been shown to exert direct lipolytic effects on coho salmon liver incubated in vitro (Sheridan and Bern, 1986). Although the RIA of SS-14 (with mammalian components) has been available for some time (cf. Taborsky and Ensinck, 1983) and appears suitable for use in fish (Eilertson, Sheridan, and Plisetskaya, unpublished data), the lack of an appropriate measure for circulating levels of the larger and more predominate form of pancreatic somatostatin in salmon has been an obstacle to the further elucidation of its function in these animals. In this paper we describe the homologous radioimmunoassay system for measuring salmon SS-25 and present some results obtained in the use of this system. MATERIALS

AND METHODS

Raising of antisera. Coho salmon (0. /hutch) sSS25 was isolated and purified from pancreatic islets collected from mature fish (Plisetskaya et al., 1986). Antibodies were raised in two three-month old white New Zealand male rabbits according to Vaitukaitis et al., 1971. Before injection, the antigen (sSS-25) was conjugated with bovine thyroglobulin (Tg, Sigma) using I-ethyl-3-(diethylaminopropyl)carbodiimide-HCL (CDI; Sigma) as a coupling agent. The molar ratio of sSS-25:Tg:CDI was 1 l&1:200; conjugation time was 12-18 hr at room temperature. For the first injection, each rabbit received intradermally 2.0 ml of mixture containing about 200 pg of conjugated sSS-25 (in 0.9 ml of 0.15 M NaCl) emulsified in 1.0 ml complete Freund’s adjuvant supplemented with 0.1 ml of dessicated Myobacterium tuberculosis (25mg/ml; DIFCO)

AND

PLISETSKAYA

and 150 pg of adjuvant peptide (Sigma). After 7, 10, and 22 weeks, each rabbit was given a booster injection containing 75 ug of conjugated sSS-25 emulsified in incomplete Freund’s adjuvant supplemented with M. tuberculosis and adjuvant peptide (150 ug). Test bleedings were performed 10 days after each booster and the antibody production was tested in an enzyme immunoassay (ELISA) for soluble antigens (Engvall, 1980). The second antibody in these assays was peroxidase-labeled goat anti-rabbit gammaglobulin. (Cappel Laboratories). Serial dilutions of antiserum (lot 3323) were tested for cross-reactivity with the sSS-25 tracer. The primary dilution of 1:7000, which appeared to bind 25-30% of tracer added (7000-8OOtl cpm), was chosen for subsequent assays. Iodination of salmon somatostatin-25. Purified coho sSS-25 was iodinated using a chloramine-T method (Odell, 1983). One millicurie of NaiZ51 in 10 pl of 1 mM NaOH (IMS-30; Amersham) and 2.5 ug of chloramineT (0.5 &ml in 0.05 M phosphate buffer containing 0.15 NaCl, pH 7.4) were added to ca. 2 ug of peptide in 50 (~1of 0.5 M phosphate buffer containing 0.15 M NaCI, pH 7.0. The reaction was stopped after 20 set by the addition of 300 ~15% (w/v) bovine serum albumin (BSA; Sigma). The label was repurified by gel filtration on a Sephadex G-25 column (1 X 50 cm) previously seasoned with 100 ml of 0.1 M acetic acid. The iodination mixture was eluted with 0.05 M sodium acetate buffer (pH 5.0) and 1.5 ml fractions were collected in tubes containing 20 ~1 of 5% BSA. The specific activity of the ‘251-sSS-25 was 50-100 uCi/ug and appeared to be stable for 1 month when stored at - 20”. The tracer was considered unusable when nonspecific binding (NSB) became greater than 10%. Radioimmunoassay procedure. A double-antibody radioimmunoassay under disequilibrium conditions, modified after Taborsky and Ensinck (1983), was used. The assay buffer was 0.01 M phosphate buffer, pH 7.4, containing 0.15 M NaCl, 0.01 M EDTA, 0.5% BSA, 0.1% gelatin, and 0.1% NaN, and 1.4% inhibitor cocktail (0.1 g/ml benzamidine, 13.4 mg/ml sodium citrate dihydrate, 17 IU/ml heparin, 0.1 mg/ml soybean trypsin inhibitor, 0.02 mg/ml o-phenanthroline, 0.04 mg/ml EDTA and 1.25 mg/ml N-ethylmaleimide). Standard (from 5 to 120 pg) or sample, in 100 p.1, was added to assay tubes prior to addition of 300 ul of assay buffer and 100 pl of antisera (initial dilution 1:7000). After 24 hr at 4”, 100 ul of ‘z51-sSS-25 (ca. 7000 cpm) was added to each tube and the incubation continued for another 24 hr. Bound hormone was sep arated from free by addition of 100 ~1 of goat antirabbit gammaglobulin (initial dilution 1:7) and 1.0 ml of polyethylene glycol (PEG)-rabbit gammaglobulin (RGG) (25 mg/ml PEG and 19 &ml RGG in assay buffer). After incubation for 2 hr at 4”, precipitate was collected by centrifugation at 2OOOgfor 30 min at 4”. Supematant was aspirated and the radioactivity in the

RIA

OF

SALMON

bound fraction (pellet) counted in a Beckman gamma counter. All values were corrected for nonspecific binding by substituting 200 ul of assay buffer (buffer blank) for the antisera and sample or 100 pl of assay buffer for the antisera and 100 ul of coho plasma for sample (plasma blank). In the course of these experiments, no significant difference was detected between the two blanks. The total radioactivity bound by the antibody in the absence of unlabeled hormone (Be/T) was 25-30%. The results were calculated using a HewlettPackard computer program which developed a linearized standard curve by log-logit transformation. Source of hormones. Mammalian insulin (bovine, I-5500), ghrcagon (bovine/porcine, G-4250) and mammalian somatostatin-28 (S-6135) were obtained from Sigma. Synthetic SS-14 and synthetic Gillichthys urotensin II were obtained from Peninsula Laboratories (San Carlos, CA). Source ofplasma, pancreatic islets, and experimental animals. Plasma from mature whitefish (Coregonus clupeaformis), lake trout (Salvelinus namaycush), arctic char (S. alpinus), and brook trout (S. fontinalis)

were generously provided by Dr. J. G. Eales (University of Manitoba, Winnipeg, MB Canada). Plasma from chinook salmon (0. tshawytscha) was obtained from spawning male fish (4-6 kg) captured by electroshocking on Lake Sakakawea (near Riverdale, ND). Plasma from juvenile coho salmon (0. kisutch) (age 1 + , both sexes) was obtained from Lake Washington strain fish maintained at the Northwest and Alaska Fisheries Research Center (Seattle, WA). Plasma from rainbow trout (0. mykiss) was obtained from juvenile (age 1 + , both sexes) animals provided by the U.S. Fish and Wildlife Service (Garrison National Hatchery, Riverdale, ND) and maintained at North Dakota State University. Plasma from various nonsalmonid species (cattish, Ictaluris natalis; sucker, Catostomus commersoni; bluegill, Lepomis macrochirus) was generously provided by Dr. J. J. Peterka (North Dakota State University). Brockmann bodies were removed from juvenile coho salmon (Northwest and Alaska Fisheries Research Center) and rainbow trout (North Dakota State University). Tissue extracts were prepared as described previously by Plisetskaya et al. (1985). Dilutions (I:3000 to 1:24,000) of the extracts were then assayed by RIA. Glucose-injection experiments were performed on juvenile chinook salmon (0. tshawytscha) maintained at North Dakota State University in flowing dechlorinated municipal water (14”) under a 12L: 12D photoperiod. Experiments were conducted on four groups of fish (8-12 individuals per group, 43-55 g) between January and April of 1989; experiments were repeated three times. Fish were fed ad libirum with Glenco Mills trout chow (Glenco, MN) except 48 hr prior to experimentation. Animals were anesthetized in buff-

367

SOMATOSTATIN

ered MS 222 (0.1%. w/v) and injected intraperitoneally (10 PI/g body wt) with 2000 mg/dl glucose made up in 0.7% NaCI. Control fish were injected with saline. Animals were sampled 30 and 60 min after injection. The fish were stunned by a sharp blow to the head, and blood was collected from the severed caudal vessels into heparinized capillary tubes. Plasma was stored at - 20” for later hormone analysis and glucose determination. Glucose was determined by the o-toluidine method described by Huvarinen and Nikkila (1962). Statistics. Results from islet hormone content and glucose loading experiments are presented as means 2 SEM and were evaluated by one-way analysis of variance (ANOVA); multiple comparison among means was made by the Student-Newman-Keul’s test. Evaluation of cross-reactivity was performed by paired analysis (t test) of regression slopes of linearized (loglogit) dose-inhibition curves. Lines with no slope (b = 0) were considered not to display cross-reactivity; lines with equal slope were considered parallel. In all cases a was set at 0.05.

RESULTS

Specificity of the antibody was initially screened by ELISA (sSS-25 antisera, initial dilution 1:5000). These experiments indicated that the antibody did not cross-react with SS-14 or with urotensin II (U II), a dodecapeptide secreted by the caudal neurosecretory system of fish. Specificity of the antibody under RIA conditions was examined by evaluating cross-reactivity with several peptides. The antiserum did not cross-react (P > 0.05) with U II, insulin, glucagon or somatostatin-28; there was negligible (0.13%; P > 0.05) crossreactivity with SS-14 (Fig. 1). The specificity of the antibody was also tested by using various dilutions of plasma from selected salmonid and nonsalmonid fish species. Figure 2 shows that parallel (P < 0.05) displacement curves were obtained with plasma from a majority of the salmonid species tested; cross-reactivity was less pronounced (and nonparallel) for whitefish, lake trout, and arctic char. Of the nonsalmonid plasma examined, significant (b > 0; P < 0.05) displacement occurred with plasma from sucker and bluegill. Significant

368

SHERIDAN,

0 0

EILERTSON,

I

I I

I ,

20

40

60

,, I,

AND PLISETSKAYA

I I

I I

,,, ,I

I I

I

300 600 3000 6000

pg SS-25/tube FIG. 1. Dose-response inhibition curves for coho somatostatin-25 (SS-29, somatostatin-14 (SS-14), mammalian somatostatin-28 (SS-28), mammalian insulin, mammalian glucagon, and urotensin II (U II). Each point is the mean of triplicate determinations.

displacement did not occur with the catfish plasma. Figure 3 illustrates the displacement curves for pancreatic extracts from some SALMONID PLASMA bl) 6.25 25 100 L b I

I

salmonid species. Serial dilution of extracts from coho salmon and rainbow trout both gave inhibition curves with slopes that were parallel to the sSS-25 standard curve. Islet

NONSALMONID PLASMA 011) 6.25 25 100 -

pancreatic 1:24000

islet extract t:1*oQ*

1:6000

dilution 13000

loo? 9 ii 2 I w 1 a?

l 8 a A\

80. \ 60.

A\ A*

l \\

40-

‘i

2 t 4 %

04

&I SS-25/tube

FIG. 2. Dose-response inhibition curves for coho salmon somatostatin (SS-25) and serial dilutions of plasma from coho and chinook salmon, rainbow, brook and lake trout. arctic char, whitefish, bluegill, sucker, and cattish. Each point represents the mean of triplicate determinations.

0

20

40

60

PLJ SS-25/tube

FIG. 3. Competitive binding curves for coho salmon somatostatin-25 standard, and serial dilutions of crude principle islet extracts from coho salmon and rainbow trout. Each point represents the mean of triplicate determinations.

RIA

OF

SALMON

somatostatin concentration was estimated to be between 8.4 -+ 0.8 (n = 4) ng SS25/mg fresh wt islet tissue for trout and 14.0 + 1.4 (n = 4) units for salmon. Salmon SS-25, as well as raw plasma and ethanol-extracted plasma, was subjected to liquid chromatography and the effluent fractions were analyzed by RIA (Fig. 4). In both raw plasma and ethanol-extracted plasma, sSS-25 immunoreactive peaks were detected in fractions corresponding to the sSS-25 standard. Recovery of sSS-25 as assessed was tested by adding increasing amounts to sSS-25 (in 10 ~1 of assay buffer) to 90 p.1 of catfish plasma (that plasma exhibiting the SS-25

STANDARD

369

SOMATOSTATIN

lowest cross-reactivity with antiserum). The recovery was 99% in six assays. The lowest detectable level of sSS-25 was 5 pgl tube (the lowest amount of hormone that can be distinguished from 0 dose and calculated as twice the standard deviation at 0 dose); especially reproducible results were obtained in the range from 0.15-1.20 r&ml (15-120 pg/tube corresponding to BIB, = 80% and BIB, = 20%, respectively) using 100 ~1 of plasma, which appears to be the normal range of sSS-25 circulating in the plasma of salmon. The coefficients of variation for a repeated set of samples were 12.6% interassay (n = 6) and 5.7% (n = 6) intraassay . The RIA system for sSS-25 was used to measure the plasma circulating levels of sSS-25 in glucose-injected salmon (Fig. 5). Glucose injection resulted in hyperglyce-

~ :;I::, -

0 I

WNE-INJECTED GLUCOSE-INJEClED

*

PLASMA

In 100 c-4 I :

NON-EXTRACTED

75-

Ol--1

W&ED

n1 I 5

9

13

17 21

FRACTION

25

29

33

37

41

45

49

NUMBER

FIG. 4. Liquid chromatography (Bio-Rad Laboratories Bio-gel A-l.5 m, 200-400 mesh; column size: 1.5 X 35 cm) of salmon somatostatin-25 standard (10 ng), raw chinook salmon plasma, and extracted (ethanol: plasma, 1.5: 1) chinook plasma. Extracted plasma was dried (Speed-vat, Savant Inc.) and reconstituted in assay buffer. One milliliter of each substance was applied to the top of the column and eluted with 0.1 M acetic acid at a flow rate of 0.86 ml/min into plastic tubes (12 x 75 mm) containing 20 ~1 inhibitor cocktail (see text) and 1S-ml fractions were collected. RIA was performed on effluent fractions as described in the text.

% Fe. * c J 8E

*

05. '

\ wm (J5

I

%? 0.0

30 MINUTES

60 AFTER

INJECTION

FIG. 5. Effects of glucose load on plasma glucose and plasma somatostatin-25 concentrations. Glucose (2000 mg/dl) was injected intraperitoneally (10 pi/g body wt) into juvenile chinook salmon and plasma was collected 30 and 60 min after injection. Data are presented as the means T SEM (n = 8-12); *Significantly different from control (P < 0.05).

370

SHERIDAN.

EILERTSON,

mia within 30 min and persisted 60 min after injection. Plasma sSS-25 concentrations followed changes in blood glucose. Plasma sSS-25 levels in glucose-injected animals were significantly elevated, both 30 and 60 min after injection, over levels observed in saline-injected controls.

AND

PLISETSKAYA

ant of somatostatin (Andrews et al., 1984). Interestingly, antiserum raised against catfish SS-22 cross-reacted very poorly with anglerfish SS-28 (Fletcher et al., 1983) as well as with SS-14 (Ronner and Scat-pa, 1987).

The anti-sSS-25 antiserum used for the RIA development was sensitive and highly specific. The antibody failed to bind with DISCUSSION other pancreatic peptides, such as insulin The assay of sSS-25 was developed in a and glucagon. Most importantly, the antifully homologous radioimmune system that body did not bind or showed negligible included sSS-25 as standard and tracer as binding with mammalian somatostatin-28 well as primary antibody raised against and SS-14. Furthermore, the antibody sSS-25 in rabbit. The only other homolofailed to bind with U II, a peptide with gous assay for a variant somatostatin form structural similarity to SS-14 (Pearson ef derived from preprosomatostatin, other al., 1980). than preprosomatostatin-I, was reported by The lack of significant cross-reactivity Oyama et al. (1982) and Fletcher et al. with SS-14 in the assay system is an impor(1983) for catfish somatostatin-22 (SS-22). tant finding, since there appears to be However, this assay has been used only for chemical and biological similarities bethe measurements of SS-22 in extracts of tween these peptides may lie in the antisevarious tissues and not for determination of rum against sSS-25, which according to SS-22 concentration in plasma. Therefore, Nozaki et al. (1988) possibly contains two the present assay of sSS-25 seems to be the immunoreactive sites: one in the Nfirst assay developed for gene-II somatoterminal and a second in the C-terminal statin circulating in fish plasma. This RIA portion of the molecule. Since the second based on coho salmon components can be somatostatin possessed by teleost fish is applied for the assessments of plasma and the invariant 14-amino acid form (SS-14) tissue contents of immunoreactive SS-25 in derived from gene-I, our novel RIA makes other salmonid and some nonsalmonid spe- it possible to resolve titers of sSS-25 from cies. For example, the displacement curves those of SS-14. This will significantly enof coho salmon (0. kisutch) plasma, as well hance our understanding of the differential as plasma from closely related species (0. effects of these peptides in salmon and fish tsawytscha, 0. my&s), are parallel to the generally. To date, few reports about the standard curve of sSS-25, indicating the differential effects of the two forms of sospecificity of RIA for plasma assays in matostatin have appeared (cf. Marchant et these species of fish. This implies that these al., 1987; Marchant and Peter, 1989). Our fish probably produce the same or similar preliminary results indicate that sSS-25 and N-terminally extended somatostatin. InSS- 14 are equipotent in their ability to stimdeed, the same antiserum that we employed ulate glycogenolysis in salmon (Eilertson in RIA immunostained D-cells in the endo- and Sheridan, 1989) or to modulate the crine pancreas (Brockmann body) of plasma levels of other pancreatic hormones (cf. Plisetskaya, 1989b). The topology of salmon, trout (Nozaki et al., 1988), and gilthead sea bream, Sparus auratus (Abad et somatostatin-producing cells in the endoal., 1989). On the other hand, the assay sys- crine pancreas of salmonids and some other tem cannot be used with cattish plasma as fish (reviewed by Nozaki et al., 1988; Plimight be expected since this species pos- setskaya, 1989b) may imply some specific sesses a very different 22-amino acid vari- interrelations between SS-14 and insulin

RIA

OF SALMON

and between SS-25 and glucagon-family peptides, respectively. These interrelations remain to be investigated. As far as we are aware, the levels of somatostatin, either the 1Camino acid form or the N-terminally extended forms, circulating in the plasma of fish have not been previously reported. The plasma levels of SS-25 measured by us appear to be 0.2-0.6 &ml, which are comparable to levels of total somatostatin-like immunoreactivity (SLI) (Taborsky and Ensinck, 1983) and higher than SS-14 levels (Ensinck et al., 1989) found to circulate in the plasma of mammals. It should be noted that these values were determined on raw plasma and may, due to interfering substances, overestimate actual levels. Although what is considered “actual” may vary according to the vascular compartment measured. Plisetskaya and Sullivan (1989) reported in trout that the concentrations of islet hormones such as insulin, glucagon, and glucagon-like peptide are significantly (ca. fivefold) higher in the hepatic portal vein compared to the caudal vasculature. Induced changes of plasma SS-25 levels after glucose injection provided further evidence that the SS-25 RIA is valid for physiological experiments. In the present experiment, elevated plasma sSS-25 concentrations were associated with hyperglycemia. This observation is consistent with the reports that plasma SLI increases following a meal (Polonsky er al., 1983) and that glucose modulates SLI release from the gut (Schmid et al., 1988). It is more controversial whether the meal also stimulates somatostatin release from the endocrine pancreas (Ipp et al., 1977) or remains a paracrine regulator within the pancreatic islets (Klaff and Taborsky, 1987). The design of our pilot experiments did not allow us to address this question. Ince and So (1984) and Ronner and Scarpa (1987) reported that in catfish and eel, glucose is a potent secretagogue of SLI release from pancreas both in viva and in situ. However, it still remains to be investigated whether in viva glucose

371

SOMATOSTATIN

affects islet N-terminally extended somatostatin secretion directly or indirectly via some other pancreatic or gut hormone(s). ACKNOWLEDGMENTS We are indebted to the North Dakota Department of Game and Fish and the Northwest and Alaska Fisheries Research Center for providing experimental animals. We also thank Drs. J. G. Eales and J. J. Peterka for providing fish plasma, P. Have1 for his help in raising antibodies, and G. J. Taborsky, Jr., and J. M. Conlin for their advice in establishing the somatostatin assay. The valuable assistance of Kim Michelsen, Jamie Harmon, and Pamela O’Connor is also gratefully acknowledged. This work was sponsored by the National Science Foundation (DCB 8901380 to M.A.S. and DCB 8615551 to E.M.P.).

REFERENCES Abad, M. E., Lozoan, M. T., Taveme-Thiele, J. J., and Rombout, J. H. W. M. (190). Identification of two somatostatin-immunoreactive cell types in the principle islet of Sparus aurufus L. (Teleostei) by immunogold staining. Gen. Cump. Endocrinol. 17, I-8.

Andrews, P. C., and Dixon, J. E. (1981). Isolation and structure of a peptide hormone predicted from a mRNA sequence: A second somatostatin from the catfish pancreas. J. Biol. Chem. 256, 826744270. Andrews, P. C., Pubols, M. N., Hermodson, M. A., Sheares, B. T., and Dixon, J. E. (1984). Structure of the 22-residue somatostatin from catfish. An O-glycosylated peptide having multiple forms. J. Biol. Chem. 259, 13,267-13,272. Argos, P., Taylor, W. L., Minth, C. D., and Dixon, J. E. (1983). Nucleotide and amino acid sequence comparisons of preprosomatostatins. J. Biol. Chem. 258, 8788-8793. Benoit, R., Bohlen, P., Brazeau, P., Ling, N., and Guillemin, R. (1980). Isolation and characterization of pancreatic somatostatin. Endocrinology 107, 2127-2129. Brazeau, P., Vale, W., Burgus, R., Ling, N., Butcher, M., Rivier, J., and Guillemin, R. (1973). A hypothalamic polypeptide that inhibits the secretion of pituitary growth hormone. Science 179, 77-79. Conlon, J. M. (1989). Biosynthesis of regulatory peptides--evolutionary aspects. In “The Comparative Endocrinology of Regulatory Peptides” (S. Holmgren, Ed), pp. 344-369. Chapman and Hall, London/New York. Eilertson, C. D., and Sheridan, M. A. (1989). Effects of somatostatin-14 and somatostatin-25 on the carbohydrate and lipid metabolism of salmonids. Amer. Zool., 29, 19A. Engvall, E. (1980). Enzyme immunoassay: ELISA and

372

SHERIDAN,

EILERTSON,

EMIT. In “Methods in Enzymology” (H. Van Vunakis and J. J. Langdone, Eds), vol 70, pp. 419-439. Academic Press, New York. Ensinck, J. W., Laschansky, E. C., Vogel, R. E., Simonowitz, D. A., Roos, B. A., and Francis, B. H. (1989). Circulating prosomatostatin-derived peptides. J. Clin. Invest. 83, 1580-1589. Fletcher, D. J., Trent, D. F., and Weir, G. C. (1983). Cattish somatostatin is unique to piscine tissues. Regul. Pept. 5, 181-187. Huvarinen, A., and Nikkila, E. A. (1%2). Specific determination of blood glucose with o-toluidine. Clinical Chim. Acta 7, 14&143. Ince, B. W., and So, S. T. C. (1984). Differential secretion of glucagon-like and somatostatin-like immunoreactivity from perfused eel pancreas in response to o-glucose. Gen. Comp. Endocrinol. 53, 389-397. Ipp, E., Dobbs, R. E., Arimura, A., Vale, W., Harris, V., and Unger, R. (1977). Release of immunoreactive somatostatin from pancreas in response to glucose, amino acids, pancreozymin-cholecystokinin, and tolbutamide. J. Clin. Invest. 60, 760765. King, J. A., and Millar, R. P. (1979). Phylogenetic and anatomical distribution of somatostatin in vertebrates. Endocrinology 105, 1322-1329. Klaff, L. G., and Taborsky, G. J., Jr. (1987). Pancreatic somatostatin as a mediator of glucagon inhibition by hyperglycemia. Diabetes 36, 592-596. Marchant, T. A., Fraser, R. A., Andrews, P. C., and Peter, R. E. (1987). The intluence of mammalian and teleost somatostatins on the secretion of growth hormone from goldfish (Carussius aurutus L.) pituitary fragments in vitro. Regul. Pep?. 17, 41-52. Marchant, T. A., and Peter, R. E. (1989). Hypothalamic peptides influencing growth hormone secretion in the goldfish, Carassius auratus. Fish Physiol. Biochem. 7, 133-139. Noe, B. D., Spiess, J., Rivier, J., and Vale, W. (1979). Isolation and characterization of somatostatin from anglerfish pancreatic islet. Endocrinology 105, 14161415. Nozaki, M., Miyata, K., Oota, Y., Gorbman, A., and Plisetskaya, E. M. (1988). Different cellular distributions of two somatostatins in brain and pancreas of salmonids and their association with insulin- and glucagon-secreting cells. Gen. Comp. Endocrinol. 69, 267-280. Odell, W. D. (1983). Radiolabeling techniques. In “Principles of Competitive Protein-Binding Assays” (W. D. Ode11and P. Franchimont, Eds.), 2nd ed. pp. 69-83. Wiley, New York. Oyama, H., Gabbay, K., Loo, S. W., Hirsch, H., Greider, M., and Permutt, A. (1982). Radioimmu-

AND PLISETSKAYA noassay for catfish pancreatic somatostatin-22. Regul. Pept. 3, 383-396. Pearson, D., Shively, J. E., Clark, B. R., Geschwind, I. I., Barkley, M., Nishioka, R. S., and Bern, H. A. (1980). Urotensin II: A somatostatin-like peptide in the caudal neurosecretory system of fishes. Proc. Natl. Acad. Sci. USA 77,5021-5024. Plisetskaya, E. M. (1989a). Pancreatic peptides. In “The Comparative Physiology of Regulatory Peptides” (S. Holmgren, Ed), pp. 174-202. Chapman and Hall, LondonMew York. Plisetskaya, E. M. (1989b). Physiology of fish endocrine pancreas. Fish Physiol. Biochem. 7, 39-48. Plisetskaya, E. M., Pollock, H. G., Rouse, J. B., Hamilton, J. W., Kimmel, J. R., Andrews, P. C., and Gorbman, A. (1986). Characterization of Coho Salmon (Oncorhynchus kisutch) islet somatostatins. Gen. Comp. Endocrinol. 63, 252-263. Plisetskaya, E., Pollock, H. G., Rouse, J. B., Hamilton, J. W., Kimmel, J. R., and Gorbman, A. (1985). Characterization of coho salmon (Oncorhynchus kisutch) insulin. Regul. Pept. 11, 105116. Plisetskaya, E. M., and Sullivan, C. V. (1989). Pancreatic and thyroid hormones in rainbow trout (Salmo gairdneri): What concentration does the liver see? Gen. Comp. Endocrinol. 75, 310-315. Polonsky, K. S., Shoelson, S. E., and Docherty, H. M. (1983). Plasma somatostatin-28 increases in response to feeding in man. J. Clin. Invest. 71, 1514-1518. Ronner, P., and Scarpa, A. (1987). Secretagogues for pancreatic hormone release in the channel catfish (Zctalurus punctatus). Gen. Comp. Endocrinol. 65, 354-362. Schmid, R., Schusdziarra, V., and Classen, M. (1988). Modulatory effect of glucose on VIP-induced gastric somatostatin release. Amer. .I. Physiol. 254, E756E759. Sheridan, M. A., and Bern, H. A. (1986). Both somatostatin and the caudal neuropeptide, urotensin II, stimulate lipid mobilization from coho salmon liver incubated in vitro. Regul. Pept. 14, 333-344. Sheridan, M. A., Plisetskaya, E. M., Bern, H. A., and Gorbman, A. (1987). Effects of somatostatin25 and urotensin II on lipid and carbohydrate metabolism of coho salmon, Oncorhynchus kisutch. Gen. Comp. Endocrinol. 66, 405414. Taborsky, G. J., Jr., and Ensinck, J. W. (1983). Extraction of somatostatin by the pancreas. Endocrinology 112, 303-307. Vaitukaitis, J., Robbins, J. B., Neischlag, E., and Ross, G. T. (1971). A method for producing specitic antisera with small doses of immunogen. J. Clin. Endocrinol. 33, 988-991.