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Endocrinology, doi:10.1210/en.2003-0439
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Endocrinology Vol. 144, No. 9 4031-4041
Copyright © 2003 by The Endocrine Society

Endogenous Hypothalamic Somatostatins Differentially Regulate Growth Hormone Secretion from Goldfish Pituitary Somatotropes in Vitro

Warren K. Yunker, Sean Smith, Chad Graves, Philip J. Davis, Surajlal Unniappan, Jean E. Rivier, Richard E. Peter and John P. Chang

Department of Biological Sciences (W.K.Y., S.S., C.G., P.J.D., S.U., R.E.P., J.P.C.), Faculty of Science, University of Alberta, Edmonton, Alberta, Canada T6G 2E9; and Faculty of Medicine and Dentistry (W.K.Y.), University of Alberta, Edmonton, Alberta, Canada T6G 2R7; and The Salk Institute (J.E.R.), La Jolla, California 92037

Address all correspondence and requests for reprints to: Dr. J. P. Chang, Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9. E-mail: john.chang{at}ualberta.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Using Southern blot analysis of RT-PCR products, mRNA for three different somatostatin (SS) precursors (PSS-I, -II, and -III), which encode for SS14, goldfish brain (gb)SS28, and [Pro2]SS14, respectively, were detected in goldfish hypothalamus. PSS-I and -II mRNA, but not PSS-III mRNA, were also detected in cultured pituitary cells. We subsequently examined the effects of the mature peptides, SS14, gbSS28, and [Pro2]SS14, on somatotrope signaling and GH secretion. The gbSS28 was more potent than either SS14 or [Pro2]SS14 in reducing basal GH release but was the least effective in reducing basal cellular cAMP. The ability of SS14, [Pro2]SS14, and gbSS28 to attenuate GH responses to GnRH were comparable. However, gbSS28 was less effective than SS14 and [Pro2]SS14 in diminishing dopamine- and pituitary adenylate cyclase-activating polypeptide-stimulated GH release, as well as GH release resulting from the activation of their underlying signaling cascades. In contrast, the actions of a different 28-amino-acid SS, mammalian SS28, were more similar to those of SS14 and [Pro2]SS14. We conclude that, in goldfish, SSs differentially couple to the intracellular cascades regulating GH secretion from pituitary somatotropes. This raises the possibility that such differences may allow for the selective regulation of various aspects of somatotrope function by different SS peptides.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE SOMATOSTATIN (SS) neuropeptide family is comprised of multiple genes and gene products. In mammals, there are two biologically active forms of SS, SS14 and its N-terminal extension mammalian (m)SS28, both of which are derived from the same precursor molecule, prosomatostatin (PSS)-I (reviewed in Ref.1). The cDNAs for PSS-I have also been cloned from numerous nonmammalian species, including chicken, frog, and several teleost fish, including anglerfish, rainbow trout, sturgeon, and goldfish (Ref.2 , reviewed in Ref.3). Similar to mPSS-I, goldfish PSS-I is capable of yielding SS14. In fact, the SS14 derived from goldfish PSS-I is identical to mSS14 in amino acid sequence (4). However, unlike mammalian PSS-I, goldfish PSS-I does not contain a monobasic Arg cleavage site capable of yielding SS28. Although there is a cleavage site capable of yielding a 26-amino-acid SS (4), a SS26 has not been isolated.

In addition to PSS-I, goldfish possess two additional PSSs, PSS-II and PSS-III. Goldfish brain (gb)SS28, which is contained within PSS-II, differs from mSS28 in two ways. First, in addition to differing by eight amino acids in the N terminus, it contains [Glu1, Tyr7, Gly10]SS14 at its C terminus (4). Second, it is a separate gene product, rather than an alternate cleavage product (4). Although goldfish PSS-II contains cleavage sites capable of generating both 14- and 28-amino-acid peptides, studies in anglerfish have shown that the 14-amino-acid SS and the 28-amino-acid SS are separate, independent products of PSS-I and PSS-II, respectively (5, 6, 7, 8). As such, it is believed that goldfish PSS-II yields only gbSS28.

The third goldfish PSS, PSS-III, contains a potential cleavage site for a 14-amino-acid peptide but not for a 28-amino-acid peptide (4). There are, however, Arg monobasic cleavage sites capable of yielding 24- and 29-amino-acid peptides. However, a [Pro2]SS14 has been identified in Russian sturgeon (9), making the occurrence of [Pro2]SS14 in goldfish a likely possibility. Phylogenetic analysis suggests that PSS-III is related to the [Pro2, Met13]SS14 precursor in frog, and cortistatin in mammals (4, 10).

In mammals, SS14 and SS28 are differentially expressed throughout the central nervous system, peripheral nervous system, and most of the major organs of the body (11, 12, 13), whereas cortistatin is expressed primarily in the cerebral cortex and hippocampus (14, 15). Similarly, in several nonmammalian vertebrates, including frog (16), coho salmon (17), rainbow trout (17, 18), sturgeon (2), and goldfish (4), differential distribution of PSS-I, PSS-II, and PSS-III have been reported.

The SSs act through a family of G protein-coupled receptors. In mammals, five SS receptor subtypes (sst1–5) have been identified. Each subtype is capable of interacting with a distinct set of intracellular signaling systems (reviewed in Ref.19). All five sst’s bind SS14 and mSS28 with high affinity; however, sst5 exhibits some selectivity for mSS28. Although all five receptor subtypes are expressed in the pituitary, sst2 and sst5 are believed to be the primary regulators of somatotrope function (20). In goldfish, 8 sst’s (gfsst1A, 1B, 2, 3A, 3B, 5A, 5B, 5C) have been cloned from brain tissues (21, 22, 23, 24). Similar to the situation in mammals, pharmacological characterization of gfsst5A has shown that although it binds SS14, [Pro2]SS14 and gbSS28 with high affinity, it displays some selectivity for 28-amino-acid SSs (22). Consistent with mammalian studies, gfsst2 and gfsst5 mRNA is predominantly expressed in the pituitary, compared with other brain regions (22, 24).

The differential expression of sst subtypes is clearly one of the means by which functional specificity is achieved in the SS/sst system. However, whether (and how) the different SS peptides contribute to the selective regulation of cell function in tissues where more than one isoform are present is poorly understood. To begin exploring this possibility, we searched for PSS mRNA in goldfish hypothalamus and pituitary. We subsequently examined the ability of the mature peptides, SS14, [Pro2]SS14, and gbSS28, to regulate basal GH secretion and cAMP production. In addition, we examined the ability of these three SS peptides to inhibit GH release stimulated by several different goldfish neuroendocrine regulators, as well as GH secretion resulting from the pharmacological activation of their respective intracellular signaling cascades. In particular, we used GnRH, which stimulates GH release in a protein kinase C (PKC)-dependent manner, and dopamine (DA) and pituitary adenylate cyclase (AC)-activating polypeptide (PACAP), which act through AC/cAMP/protein kinase A (PKA)-sensitive mechanisms (reviewed in Ref.25). The concept of SS isoform functional selectivity was further tested by comparing the apparent intracellular mechanisms mediating mSS28 and gbSS28 inhibition of GH release.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and cell preparation
All animal maintenance and experimental protocols used in this study were approved by the University of Alberta, Biological Sciences Animal Care Committee in accordance with national guidelines. Common goldfish (Carassius auratus, 8–13 cm in length) were purchased from Aquatic Imports (Calgary, Alberta, Canada) and maintained in flow-through aquaria (1800 liters) at 16–20 C as previously described (26). As needed, goldfish were anesthetized in 0.05% tricaine methanesulfonate and euthanized by cervical transection. Pituitaries were subsequently excised and dispersed using a previously described trypsin/DNase dispersion protocol (26).

Pituitaries from male and female goldfish at different stages of the reproductive cycle were used in this study. The magnitude of SS14 inhibition of basal GH release has previously been reported to vary throughout the seasonal reproductive cycle (27). However, when all available data, collected over the last 4 yr, concerning SS14 regulation of basal GH release were pooled and analyzed according to the time of year, these changes were not significant (Yunker, W. K., unpublished data). In the present study, SS14, [Pro2]SS14, and gbSS28 inhibition of basal GH release was always significant, regardless of gonadal state. Nevertheless, where possible, the effects of the different SS peptides, either alone or against a specific secretagogue, were compared simultaneously to control for possible seasonal variation.

Reagents and test substances
All media contained M-199 (Invitrogen, Burlington, ON or Sigma-Aldrich, St. Louis, MO) with 0.1 g/liter L-glutamine, 26 mM NaHCO3, 25 mM HEPES, 100 mg/liter streptomycin, and 100,000 U/liter penicillin (pH adjusted to 7.2). Dispersion media contained Hanks’ salts and 0.3% BSA (fraction V, Calbiochem, San Diego, CA). Plating media (for overnight incubation) contained Earle’s salts and 1% horse serum (Invitrogen). Testing media was the same as dispersion media except that BSA was reduced to 0.1%. In instances in which cells were depolarized with 30 mM KCl, equimolar substitution of KCl for NaCl was employed to maintain osmolarity.

Distilled, deionized water was used to prepare stock solutions of [Pro2]SS14, gbSS28 (synthesized by Dr. J. Rivier), SS14, mSS28, mPACAP38, salmon (s)GnRH ([Trp7, Leu8]GnRH), chicken (c)GnRH-II ([His5, Trp7, Tyr8]GnRH, Peninsula Laboratories, Belmont, CA), and CsCl. 8-bromo-cAMP (8Br-cAMP), forskolin, (±)-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrochloride (SKF-38393), tetradecanoyl phorbol acetate (TPA, Research Biochemicals Inc., Natick, MA), dioctanoyl glycerol (DiC8), and A23187 (Calbiochem) were dissolved in dimethyl sulfoxide. Ionomycin (Calbiochem) and arachidonic acid (AA, Sigma-Aldrich) were dissolved in ethanol. Sodium nitroprusside (SNP, Calbiochem) was dissolved in testing medium immediately before use. Aliquots of concentrated stock solutions were stored at either room temperature or -20 C. Final concentrations were achieved by dilution in testing medium. Final concentrations of dimethyl sulfoxide and ethanol never exceeded 0.1% and had no effect on basal GH release (28), [Ca2+]i (29), or ionic currents in identified goldfish somatotropes (30).

Trizol reagent, Taq DNA polymerase, and SuperScript II RNase H- reverse transcriptase were purchased from Invitrogen. Hybond nylon membrane, Rediprime II random prime labeling system, and [{alpha}-32P]deoxy-CTP were purchased from Amersham Biosciences (Buckinghamshire, UK), and the QIAquick nucleotide removal kit was obtained from QIAGEN (Mississauga, ON).

RT-PCR and Southern blot analysis
Total RNA was extracted from freshly excised hypothalamus and pituitary, as well as dispersed pituitary cells that had been cultured overnight, using Trizol RNA isolation reagent (Invitrogen). Total RNA was reverse transcribed into cDNA using SuperScript II RNase H- reverse transcriptase (Invitrogen). PCR amplifications were carried out using primers specific for PSS-I, -II, and -III mRNA (4). The primer sets were:

SS1-F2 (5'-GCGTATCCAGTGCGCACTGGC-3') and SS1-R2 (5'-GTGAAAGTTTTCCAGAAGAA-3') for PSS-I mRNA, SS2-F1 (5'-CGAATCACAGCTACAAAGAGTC-3') and SS2-R1 (5'-CAAGCGAGGGCCTGAGCAGG-3') for PSS-II mRNA, SS3-F1 (5'-GGAGCTACAAGACTTCAAC-3') and SS3-R1 (5'-CTGTGTCAGAGTAAGTCCACG-3') for PSS-III mRNA.

PCR conditions were denaturation for 1 min at 95 C, annealing for 1 min at either 51 C for SS14 or 54 C for SS28 and [Pro2]SS14, and extension at 73 C for 1 min for a total of 30 cycles, with a final extension of 5 min at 73 C (4). The reactions were then electrophoresed on 1% agarose gels, transferred to Hybond nylon membranes (Amersham Biosciences) by capillary transfer and fixed by baking at 80 C for 2 h. The membranes were prehybridized at 65 C for 1 h in a hybridization solution containing 0.5 M NaHPO4 (pH 7.2), 7% SDS, 1 mM EDTA (pH 8.0), and 1% BSA. The membranes were then transferred into fresh hybridization solution to which [{alpha}-32P]deoxy-CTP-labeled probe was added. Probes for SS14, [Pro2]SS14, and gbSS28 were labeled using the Rediprime II random prime labeling system (Amersham Biosciences) and purified using the QIAquick nucleotide removal kit (QIAGEN) according to the manufacturer’s instructions. Hybridization was carried out overnight at 65 C. The membranes were subsequently washed twice with wash solution containing 0.04 M NaHPO4 (pH 7.2), 1% SDS, 1 mM EDTA (pH 8.0) at 65 C and exposed to a Phosphorscreen (Molecular Dynamics, Sunnyvale, CA) for 1 h. The screen was scanned using a PhosphorImager 445 SI (Molecular Dynamics) and analyzed using the IMAGEQUANT software (Molecular Dynamics). As a negative control, PCRs were performed in the absence of cDNA to examine cross-contamination of samples. As an internal control of the reverse transcription step, PCR amplification was carried out for 35 cycles of 94 C for 1 min, 50 C for 1 min, and 73 C for 1 min with primers designed on the basis of ß-actin partial cDNA sequence in goldfish (31) (unpublished sequence, GenBank accession no. AF079831).

Static incubation experiments assessing GH release
Following dispersion, cells were plated at a density of 0.25 x 106/well in 24-well plates (Falcon Primaria, Becton Dickinson Labware, Franklin Lakes, NJ) and cultured overnight at 28 C, 5% CO2 and saturated humidity (26). The next day, following a rinse in testing media, cells were cultured in the presence of natural ligands and/or pharmacological agents for 2 h (26). Experiments were performed in either triplicate or quadruplicate on each plate, and each experiment was repeated a minimum of three times, using different cell preparations each time. The testing media was subsequently removed and stored at -26 C until GH content was measured using a previously validated RIA (32). Hormone release (nanograms per milliliter) was normalized as a percentage of the mean basal control value (average = 969 ± 65.8 ng/ml) and compared using ANOVA followed by least significant difference multiple comparisons. Differences were considered significant when P < 0.05. All secretagogues employed in this study caused significantly elevations in GH release relative to basal control values. Hormone release results are presented as mean ± SEM. Regression lines and IC50s were calculated using SigmaPlot version 7.0 (SPSS Inc., Chicago, IL).

Static incubation experiments assessing cAMP
Freshly dispersed pituitary cells were plated and cultured overnight using the same procedure as described above. The next day, cells were washed with clear testing media (testing media without phenol red) and subsequently cultured for 2 h in clear testing media supplemented with varying concentrations of one of the three different SS isoforms. Experiments were performed in triplicate on each incubation plate, and each experiment was repeated a minimum of three times, using different cell preparations each time. Following drug treatment, 800 µl of clear testing media were collected to assess cAMP release, and cellular cAMP was extracted by lysing the cells with 1 ml distilled, deionized water and subsequent 30-sec sonication. All samples, released and cellular, were placed in a boiling water bath for 10 min to denature phosphodiesterases. Samples were then acetylated and assayed for cAMP content using a cAMP enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI). To facilitate pooling of data from replicate experiments, cAMP levels (picomoles per milliliter) were normalized as a percent of the mean basal control value (average = 6.58 ± .77 pmol/ml for released and 0.15 ±.01 pmol/ml for cell content) and compared using ANOVA followed by least significant difference multiple comparisons. Differences were considered significant when P < 0.05. Results are presented as mean ± SEM. Regression lines were calculated using SigmaPlot version 7.0 (SPSS Inc.).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of PSS-I, PSS-II, and PSS-III mRNA in the hypothalamus and pituitary
To assess whether pituitary cells may be exposed to SS14, [Pro2]SS14, and gbSS28, we examined the expression of their prohormone mRNA in the hypothalamus and pituitary. Because the hypothalamic neurons that directly innervate the teleost pars distalis (33) are removed by trypsin/DNase dispersion (26), the inclusion of both pituitary fragments and primary pituitary cell cultures enabled us to examine PSS mRNA expression in pituitary preparations that still contained hypothalamic nerve terminals, and preparations devoid of such terminals. The mRNA for all three PSSs was detected in the hypothalamus following Southern blot analysis of RT-PCR products (Fig. 1Go). Interestingly, although PSS-I and PSS-II mRNA were also present in dispersed pituitary cells, no PSS mRNA was detected in pituitary fragments (Fig. 1Go).



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  		FIG. 1. Southern blot analysis (below) of RT-PCR (above) for PSS-I, -II, and -III mRNA. The different lanes represent the following: 1 and 2, dispersed pituitary cells; 3, hypothalamus; 4, pituitary fragments; 5, negative control. PCR for goldfish ß-actin was used as an internal control for the reverse transcription step.

 
SS14, [Pro2]SS14 and gbSS28 on basal GH release
Previously, SS14 has been shown to dose-dependently reduce GH release from both goldfish pituitary fragments and dispersed pituitary cell preparations (27, 34); similarly, [Pro2]SS14 has been shown to reduce basal GH release from pituitary fragments (4). However, results obtained from pituitary fragment preparations may be complicated by SS acting on hypothalamic neuronal terminals present within the anterior pituitary (33). Here we demonstrate that all three SS isoforms can dose-dependently diminish basal GH release by acting at the level of the pituitary cell (Fig. 2AGo). SS14 and [Pro2]SS14 produced similar dose-response curves that were markedly different from that of gbSS28. IC50 estimates establish that gbSS28 is more potent than either SS14 or [Pro2]SS14 in reducing basal GH release (Table 1Go). Maximal inhibition, as calculated by regression analysis, was 71.90 ± 5.06% for SS14, 72.73 ± 0.46% for [Pro2]SS14, and 53.24 ± 6.18% for gbSS28.



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  		FIG. 2. Dose-dependent actions of SS14, [Pro2]SS14, and gbSS28 on basal GH secretion (A), intracellular cAMP (B), and released cAMP (C).

 

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  		TABLE 1. Summary of the effects of four different SS isoforms on basal and stimulated GH secretion from primary cultures of dispersed goldfish pituitary cells

 
SS14, [Pro2]SS14 and gbSS28 actions on cAMP and cAMP/PKA-dependent GH secretion
In goldfish, as in other vertebrate systems, cAMP/PKA-dependent signaling cascades have been implicated in the regulation of basal GH secretion (35, 36, 37). Here we examined the ability of SS14, [Pro2]SS14, and gbSS28 to modulate both released and intracellular basal cAMP levels in naïve pituitary cells. All three isoforms significantly reduced intracellular cAMP levels. However, although SS14 and [Pro2]SS14 were equal in their ability to maximally reduce intracellular cAMP levels, gbSS28 was significantly less effective (Fig. 2BGo). In contrast, none of the three SS isoforms tested significantly altered the levels of released cAMP (Fig. 2CGo). Significant reductions in basal GH release (Fig. 2AGo) did not directly correlate with significant decreases in either intra- or extracellular cAMP levels (Fig. 2Go, B and C).

We subsequently examined the ability of maximally effective concentrations of [Pro2]SS14 (100 nM; Fig. 2AGo) and gbSS28 (10 nM; Fig. 2AGo) to affect cAMP/PKA-dependent GH secretion, and compared these findings with those previously observed with a maximally effective concentration of SS14 (1 µM; Table 1Go) (27, 38). Activation of PACAP and DA-D1 receptors have been shown to stimulate GH secretion from goldfish pituitary cells through cAMP/PKA-dependent mechanisms (reviewed in Ref.25). Accordingly, treatments with maximal stimulatory concentrations of the DA D1-agonist SKF-38393 (1 µM) (28) and PACAP (10 nM) (39) significantly elevated GH release in this study (Fig. 3AGo). Similarly, direct activation of the cAMP/PKA cascade with the adenylate cyclase activator forskolin (1 µM) and the cell permeant cAMP analog 8Br-cAMP (1 mM) resulted in significant increases in GH secretion (Fig. 3AGo). As had been demonstrated for SS14 (27), [Pro2]SS14 abolished the ability of PACAP, SKF-38393, forskolin, and 8Br-cAMP to induce GH release (i.e. GH responses to treatment with SS plus secretagogue were not significantly different from responses to SS alone; Fig. 3AGo). In contrast, gbSS28 only partially reduced the GH responses to SKF-38393 and PACAP and did not alter GH responses to forskolin or 8Br-cAMP (Fig. 3BGo). These results establish that gbSS28, unlike SS14 and [Pro2]SS14, does not act distal to cAMP formation to suppress GH responses. To further test this hypothesis, we examined the ability of all three SSs to inhibit AA-stimulated GH release. Previous work from this laboratory has shown that AA mediates DA-stimulated GH secretion subsequent to cAMP formation (40). Although both SS14 and [Pro2]SS14 inhibited GH responses to 50 µM AA (Fig. 4Go, A and B), gbSS28 had no effect (Fig. 4CGo). These data support the hypothesis that these three goldfish SSs differ in their ability to act subsequent to cAMP formation to inhibit GH secretion.



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  		FIG. 3. Effects of [Pro2]SS14 (A) and gbSS28 (B) on PACAP-, DA-D1-, and 8Br-cAMP-stimulated GH release. An asterisk (*) represents a significant reduction in GH release, compared with the non-SS- exposed column of the pair. A number sign (#) represents a significant difference, compared with the SS-treated control.

 


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  		FIG. 4. Effects of SS14 (A), [Pro2]SS14 (B), and gbSS28 (C) on AA- and SNP-stimulated GH release. An asterisk (*) represents a significant reduction in GH release, compared with the non-SS-exposed column of the pair. A number sign (#) represents a significant difference, compared with the SS-treated control.

 
SS14, [Pro2]SS14, and gbSS28 on PKC-dependent GH secretion
In goldfish, PKC-dependent transduction mechanisms act independently of the cAMP/PKA pathway to stimulate GH secretion. Two endogenous GnRHs, sGnRH and cGnRH-II, have been shown to evoke GH release through activation of PKC, and SS14 is known to inhibit GH responses to PKC activation (reviewed in Ref.25). To compare the ability of different SSs to affect PKC-dependent GH release, the effects of [Pro2]SS14 and gbSS28 on GH responses to maximal stimulatory concentrations of sGnRH (100 mM) (26), cGnRH-II (100 mM) (26), and two PKC activators, TPA (100 nM) (41) and DiC8 (100 µM) (41) were examined. In the presence of 100 nM [Pro2]SS14 or 10 nM gbSS28, the GH-releasing ability of both cGnRH-II and sGnRH were completely abolished, and the responsiveness to DiC8 was significantly reduced (Fig. 5AGo). These observations are similar to those previously observed with SS14 (27) (Table 1Go). GH responses to TPA were also greatly reduced by [Pro2]SS14 (Fig. 5AGo), as was reported for SS14 (Table 1Go). In contrast, gbSS28 was completely ineffective against TPA-stimulated GH release (Fig. 5BGo). These observations reveal a surprising difference in the ability of these three goldfish SSs to affect TPA-sensitive but not DiC8-induced GH secretion.



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  		FIG. 5. Effects of [Pro2]SS14 (A) and gbSS28 (B) on GnRH- and PKC-stimulated GH release. An asterisk (*) represents a significant reduction in GH release, compared with the non-SS-exposed column of the pair. A number sign (#) represents a significant difference, compared with the SS-treated control.

 
SSs and their effects on nitric oxide-mediated GH release
Nitric oxide (NO) signaling has been implicated in both the GnRH and DA signaling pathways regulating GH secretion in goldfish (42, 43). Consequently, we examined the effectiveness of the goldfish SSs against NO-stimulated GH release, using maximally effective concentrations of SNP (100 µM) (42), an NO donor. In this study, SNP significantly increased GH release, and this stimulation was abolished by both SS14 and [Pro2]SS14 (Fig. 4Go, A and B). However, gbSS28 had no effect on SNP-stimulated GH secretion (Fig. 4CGo).

[Pro2]SS14 and gbSS28 actions on Ca2+-stimulated GH secretion
Ca2+ mobilization, from both intracellular and extracellular sources, is a component of both GnRH- and DA-stimulated GH release (37, 44). Previously, we have shown that SS14 is capable of inhibiting Ca2+-ionophore-stimulated GH release (45) (Table 1Go). Here we compared the abilities of three goldfish SSs to affect GH responses to elevations of [Ca2+]i. This was achieved through the use of the Ca2+ ionophores A23187 (10 µM) and ionomycin (10 µM), both of which increased [Ca2+]i in goldfish pituitary cells in previous experiments (46) and significantly increased GH secretion in this study (Fig. 6Go). Coincubation with [Pro2]SS14 significantly inhibited both A23187- and ionomycin-stimulated GH release (Fig. 6AGo). In contrast, gbSS28 significantly inhibited ionomycin-evoked GH release but did not affect A23817-stimulated GH release (Fig. 6BGo). Treatment with a depolarizing concentration of KCl (30 mM) has been shown to increase [Ca2+]i (47). Here this treatment significantly increased GH secretion (Fig. 7Go). All three goldfish SSs completely abolished 30 mM KCl-stimulated GH release (Fig. 7Go). These data show that apart from one exception (i.e. gbSS28 on A23187) all three SSs are able to affect GH responses to elevated [Ca2+]i.



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  		FIG. 6. Effects of [Pro2]SS14 (A) and gbSS28 (B) on Ca2+ ionophore-stimulated GH release. An asterisk (*) represents a significant reduction in GH release, compared with the non-SS-exposed column of the pair. A number sign (#) represents a significant difference, compared with the SS-treated control.

 


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  		FIG. 7. Effects of SS14 (A), [Pro2]SS14 (B), and gbSS28 (C) on 30 mM KCl -stimulated GH release. An asterisk (*) represents a significant reduction in GH release, compared with the non-SS-exposed column of the pair.

 
K+ channel involvement in mediating SS actions on basal GH release
Results from ovine somatotropes have demonstrated the importance of inwardly rectifying K+ channels (Kir) in regulating GH secretion (48). Furthermore, in rat somatotropes, SS14 has been shown to activate Kir as a means of reducing basal GH secretion (49). Because Kir channels display a particularly high affinity for monovalent and divalent cations (50), we examined Kir involvement in SS14, [Pro2]SS14, and gbSS28 inhibition of basal GH secretion using 5 mM extracellular CsCl. Application of CsCl had no effect on basal GH release and did not affect the ability of either SS14 or [Pro2]SS14 to significantly inhibit GH release (Fig. 8Go). However, gbSS28 was unable to inhibit GH secretion in the presence of CsCl (Fig. 8Go). These data suggest that SS28, but not SS14 and [Pro2]SS14, rely on Kir channels to inhibit GH release in this system.



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  		FIG. 8. Effects of 5 mM extracellular CsCl on SS14 (A), [Pro2]SS14 (B), and gbSS28 (C) inhibition of basal GH secretion. An asterisk (*) represents a significant reduction in GH release, compared with the non-SS-exposed column of the pair. A number sign (#) represents a significant difference, compared with the SS-treated control.

 
Mammalian SS28 differs from gbSS28 in its ability to inhibit GH release
The ability of mSS28 to inhibit GH secretion in goldfish has been previously shown (34). However, whether the mechanisms employed by mSS28 to modulate GH release differ from those activated by gbSS28 has not been considered. Using mSS28 at the same concentration as gbSS28 (10 nM) and the secretagogues at the same concentrations as above, we examined the ability of mSS28 to act on both the PACAP/DA and GnRH signaling cascades. The mSS28 abolished the GH responses to PACAP, SKF-38393, sGnRH, cGnRH-II, and SNP and reduced those induced by forskolin, 8Br-cAMP, DiC8, TPA, and AA (Fig. 9Go). This profile of inhibition differed noticeably from that of gbSS28 (Table 1Go), particularly in terms of effects on forskolin-, 8Br-cAMP-, SNP-, AA-, and TPA-induced secretion. These results demonstrate that despite identical concentrations, gbSS28 and mSS28 differ in the mechanisms underlying their inhibition of somatotrope GH release.



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  		FIG. 9. Effects of mSS28 on GH release evoked by SKF-38393 and mPACAP38 (A), forskolin and 8Br-cAMP (B), SNP and AA (C), sGnRH and cGnRH-II (D), and DiC8 and TPA (E). An asterisk (*) represents a significant reduction in GH release, compared with the non-SS-exposed column of the pair. A number sign (#) represents a significant difference, compared with the SS-treated control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we provide evidence that, in goldfish, pituitary cells may be exposed to multiple SS isoforms. Furthermore, we demonstrate that three different hypothalamic SSs, SS14, [Pro2]SS14, and gbSS28, differentially regulate GH secretion. Lastly, we establish that the goldfish somatotrope is capable of differentiating between endogenous gbSS28 and mSS28.

Exposure of pituitary cells to multiple SS isoforms
As had been previously shown in goldfish of unspecified sexual states (4), we demonstrate in this study the existence of three different PSS mRNAs in hypothalami obtained from sexually regressed goldfish. Therefore, it is conceivable that the mature peptides, SS14, gbSS28, and [Pro2]SS14, are participating in the neuroendocrine regulation of goldfish pituitary function. In addition, we also identified mRNA for PSS-I and PSS-II within preparations of dispersed pituitary cells. This suggests that SS14 and gbSS28 may also be produced locally at the level of the pituitary. Synthesis of hypothalamic neuropeptides within the pituitary has been well documented (reviewed in Ref.51). Immunoreactivity and/or mRNA for vasoactive intestinal polypeptide (52, 53), GnRH (54, 55), TRH (54, 56), and SS (57, 58) have all been found within mammalian anterior pituitary tissues. In addition, [Pro2, Met13]SS14 synthesis has been localized to melanotropes within the intermediate lobe of the frog pituitary (59), and [Pro2]SS14 has been purified from the pituitary of the Russian sturgeon (9). Although we have not localized the PSS mRNA to a specific pituitary lobe or cell type, the occurrence of PSS mRNA within dispersed cells suggests that local, pituitary level peptide production may be occurring. In combination with the observed expression of PSS-I, -II, and -III mRNA in the hypothalamus, it is plausible that the different SS peptides participate in the neuroendocrine, as well as paracrine and/or autocrine, regulation of goldfish pituitary physiology.

However, if hypophyseal SS synthesis were occurring, PSS mRNA should have also been detected within pituitary fragments. Although it is conceivable that the presence of other tissues within the pituitary fragments diluted the level of PSS mRNA transcripts, at least two other explanations present themselves as more likely alternatives. PSS-I, -II, and -III mRNA levels in the forebrain have been shown to vary seasonally and to differ between males and females (4). Furthermore, studies have demonstrated that, in the forebrains of both male and female fish, PSS-I and PSS-III expression is increased by estradiol (60). In the present study, pituitary fragment cDNA was made from pooled tissues collected from sexually regressed males and females (July) although dispersed pituitary cell cDNA was made from pooled tissues collected from sexually recrudescing males and females (November). As such, it is plausible that the absence of PSS mRNA transcripts within the pituitary fragments was the result of low steroid levels. Interestingly, in a previous study, PSS-I mRNA, but not PSS-II or -III mRNA, was detected within goldfish pituitary fragments by Northern blot analysis (4). Unfortunately, the gonadal stage of the fish used was not reported. Nevertheless, the hypothesis that SS peptides are being synthesized within pituitary cells and undergoing seasonal, sex steroid-dependent regulation, is consistent with this previous report.

It is also conceivable that the reason PSS mRNA was detected in cultured pituitary cells, and not freshly excised pituitaries fragments, was because the PSS-I and -II genes were transcribed only after the pituitary cells were deprived of hypothalamic influences. It should be noted that PSS-I and -II mRNA within cultured pituitary cells could not be visualized by ethidium bromide staining, even after 30 cycles of PCR. This indicates that the PSS mRNA levels within the pituitary cells were quite low. Such a finding is consistent with the possibility that PSS gene transcription commenced during overnight culture. The possibility that the removal of hypothalamic influences up-regulates the transcription of genes encoding for hypothalamic neuropeptides within pituitary cells in culture is currently being investigated in our laboratory. However, PSS-I mRNA has been previously detected within freshly excised pituitary fragments (4). Thus, it seems unlikely that PSS transcription following removal of hypothalamic innervation is solely responsible for the disparity between the results obtained from dispersed cells and pituitary fragments in the current study. Regardless of whether steroid and/or removal of hypothalamic influences modulate pituitary PSS mRNA expression, the data presented here are strongly suggestive of in vivo pituitary level peptide production. Future studies on SS release by, and/or immunocytochemical localization of SS in, dispersed pituitary cells would be an interesting test of this hypothesis.

SS14, [Pro2]SS14, and gbSS28 differentially affect GH secretion
In this study we demonstrate that three different hypothalamic SSs differ in their ability to alter GH release (Table 1Go). Of the SSs, gbSS28 was a more potent inhibitor of basal GH secretion than either SS14 or [Pro2]SS14. In addition, results with CsCl suggest that Kir channels participate in mediating gbSS28, but not SS14 and [Pro2]SS14, inhibition of basal GH secretion. The gbSS28 also differs from SS14 and [Pro2]SS14 in its ability to alter stimulated GH secretion. For example, the differential ability of these three goldfish SSs to inhibit forskolin-, 8Br-cAMP-, and AA-induced GH secretion suggests that the 14-amino-acid SSs are able to act subsequent to cAMP formation to inhibit GH release, but gbSS28 does not. Furthermore, gbSS28 differed from SS14 and [Pro2]SS14 in that it was not as effective at inhibiting GH release resulting from the activation of PKC or liberation of NO. The ability of gbSS28 to affect Ca2+-ionophore-induced, as well as DA-D1- and PACAP-stimulated, GH release also differed from that of SS14 and [Pro2]SS14. Overall, SS14 and [Pro2]SS14 are very similar in terms of their spectrum of activity; however, their activity differs markedly from that of gbSS28. Although we cannot yet conclude from these findings that the SSs are differentially regulating GH secretion or any other cellular functions in a physiologically relevant manner, differences in intracellular signaling, such as these, would be requisite.

It is also apparent that the goldfish GH secretion system not only differentiates among the three endogenous hypothalamic SSs, but also mSS28. Unlike gbSS28, mSS28 was able to inhibit forskolin-, 8Br-cAMP-, AA-, SNP-, and TPA-induced GH secretion, as well as abolish GnRH-evoked responses, when used at the same dose. These characteristics of mSS28 action resemble those of SS14 and [Pro2]SS14. This is not surprising given that the C terminus of mSS28 is identical to that of SS14.

Consistent with a previous study in frogs demonstrating the ability of SS14 and [Pro2, Met13]SS14 to regulate basal cAMP formation (61), all three goldfish SS peptides suppressed basal cAMP production. However, our results also demonstrate that whereas gbSS28 was the most potent inhibitor of GH release, it was the least effective at lowering cellular cAMP levels. This provides further evidence to support the idea that SS14, [Pro2]SS14, and gbSS28 differentially couple to intracellular effector systems. Although we cannot exclude the possibility that the sensitivity of the cAMP assay did not allow us to properly examine the relationships between declining cAMP levels and a reduction in basal GH release, our data suggest that these two events are not tightly coupled. This is consistent with results in rat showing that SS14 lowers basal GH release without altering intracellular cAMP levels and blocks GHRH-stimulated GH release while only partially attenuating cAMP production (62).

The cellular mechanisms responsible for the differences in SS function presented here are not known. However, it seems likely that the different sst subtypes are involved. In mammals, each sst subtype couples to a distinct set of intracellular signaling pathways, and although all five receptor subtypes bind SS14 and mSS28 with high affinity, sst5 does exhibit selectivity for mSS28 (reviewed in Ref.19). Similarly, characterization of goldfish sst5A revealed that although it binds all three endogenous goldfish brain SS ligands, it displays selectivity for the 28-amino-acid SSs (22). In addition, gfsst2 can be differentially activated by these three goldfish SSs. In COS-7 cells expressing gfsst2, SS14 and [Pro2]SS14, but not gbSS28, are able to inhibit forskolin-stimulated cAMP formation (24). Interestingly, gfsst2 and gfsst5 mRNA are predominantly expressed in the pituitary, compared with other brain regions (22, 24), and mammalian studies have shown that sst2 and sst5 are the primary regulators of somatotrope function (20). Thus, it is conceivable that gbSS28 is acting mainly through gfsst5, whereas SS14 and [Pro2]SS14 are acting more through gfsst2. We have begun to test this hypothesis and are currently evaluating gfsst subtype-specific actions in primary cultures of goldfish pituitary cells using nonpeptidyl sst-selective agonists.

However, this hypothesis cannot explain the differences in mSS28 and gbSS28 activity observed in the present study. Because gfsst5 binds with, and can be activated by, both mSS28 and gbSS28 with similar affinity (63), differences in the ability of these two 28-amino-acid SSs to affect GH secretion may be mediated through sst2. This remains a speculation at present because nothing is known regarding the ability of mSS28 to bind to and activate gfsst2. Nevertheless, the presence of the SS14 sequence in the C terminus of mSS28 is consistent with such a hypothesis.

Summary
Regardless of the mechanisms responsible for the differences in SS action presented, these differences have some very interesting physiological implications. In goldfish, GH secretion is regulated by a variety of neuropeptides and hypothalamic factors, some of which, stimulate GH release through different intracellular mechanisms. For example, sGnRH and cGnRH-II signaling is mediated by intracellular Ca2+ stores, extracellular Ca2+ entry, and PKC (64). In contrast, DA and PACAP stimulate GH secretion through AC/cAMP/PKA-sensitive mechanisms (reviewed in Ref.25). Furthermore, our laboratory has also shown that, in pituitary cells, the Ca2+ stores regulating hormone mRNA levels as well as hormone secretion, storage, and production are different (65, 66). The result is a system wherein ligands employ distinct signaling cascades to affect not only GH release but also the steps involved in hormone synthesis. Given the differences in SS14, [Pro2]SS14, and gbSS28 action presented here (Table 1Go), it seems likely that, in vivo, they are responsible for regulating different aspects of cell function.


   Footnotes
 
This work was supported by Natural Sciences and Engineering Research Council of Canada (NSERC) operating grants (to R.E.P. and J.P.C.) and NIH Grant DK50124 (to J.E.R.). W.K.Y. was supported by an NSERC Post-Graduate Studentship and an Alberta Heritage Foundation for Medical Research (AHFMR) MD/PhD studentship. P.J.D. was supported by an AHFMR summer studentship.

Abbreviations: AA, Arachidonic acid; AC, adenylate cyclase; 8Br-cAMP, 8-bromo-cAMP; c, chicken; DA, dopamine; DiC8, dioctanoyl glycerol; gb, goldfish brain; Kir, inwardly rectifying K+ channel; m, mammalian; NO, nitric oxide; PACAP, pituitary adenylate cyclase-activating polypeptide; PKA, protein kinase A; PKC, protein kinase C; PSS, prosomatostatin; s, salmon; SKF-38393, (±)-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrochloride; SNP, sodium nitroprusside; SS, somatostatin; sst, SS receptor; TPA, tetradecanoyl phorbol acetate.

Received April 8, 2003.

Accepted for publication May 23, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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