Somatostatin and somatostatin receptors in fish growth

Multiple forms of somatostatin (SS) and SS receptors (SSTR) are produced widely in the tissues of fish and interact to coordinate numerous physiological processes. Insight into their role in growth regulation emerged from studies of abnormal growth and of whole animals. The influence of SS on organismal growth operates at several levels of the growth hormone (GH)-insulin-like growth factor-1 (IGF-1) system. SS inhibits production and release of pituitary GH, but not all forms of SS are equipotent in this action. SS also influences the GH-IGF-1 system in an extrapituitary manner by reducing sensitivity to GH as well as by inhibiting IGF-1 production and secretion, and diminishing IGF-1 sensitivity. Peripheral actions of SS are important for the local control of growth and may help to coordinate growth with other processes such as metabolism, development, and reproduction by reprogramming cell responsiveness.     

Preparation of 3H-[3-M3-His2]TRH as an improved ligand for TRH receptors

[3H]-3-methyl-His2]thyrotropin releasing hormone ([3H]MeTRH) binds to sites in the sheep anterior pituitary gland which appear to be the same as those occupied by [3H]TRH and which can therefore be identified as TRH receptors. In competition experiments performed in parallel, both ligands gave the same number of binding sites, 15 pmol/g wet weight, and showed the same pharmacology for 19 TRH analogs ranging over more than 5 orders of magnitude in potency. The apparent dissociation constant of binding of [3H]MeTRH was about 3.5 nM compared to 29 nM for [3H]TRH. Kinetic experiments with [3H]MeTRH yielded a rate constant for association of 1.4 x 10(7) M-1 min-1 and rate constants for the biphasic dissociation of 5 x 10(-2) min-1 (fast phase) and 7 x 10(-3) min-1 (slow phase). Detailed methods are described for preparation of [3H]MeTRH by reduction of the dehydroproline precursor and its purification by ion exchange and antibody affinity chromatography. The major advantage of the new ligand is that its higher affinity of binding gives relatively less non-specific binding than is obtained with [3H]TRH, particularly in central nervous tissue.     

Glucocorticoid receptors in the gill tissue of fish

The presence of glucocorticoid-binding macromolecular receptors was demonstrated in the Na₂MO₄ (10 mM)-stabilized gill cytosol of the American eel, Anguilla rostrata and in that of the trout, Salmo gairdneri. In all experiments, tritiated triamcinolone acetonide ([³H]TA) was used as ligand. In the eel, the steroid was bound with a K_D of 2.84 ± 0.4 nM and an N_max of 188 ± 34 fmol/mg protein. The binding parameters for the trout cytosol were K_D = 1.43 ± 0.13 nM; N_max = 271 ± 113 fmol/mg protein. Competition studies with [³H]TA-labeled eel gill cytosol and radioinert steroids gave the following binding hierarchy: TA > dexamethasone > cortisol > 11-deoxycortisol > 21-deoxycortisol. Aldosterone, estrogens, or androgens did not complete. The eel gill receptor was deactivated by prior treatment with trypsin or mersalyl. RNase was without effect, but DNase degraded the receptor except when used in the presence of trypsin inhibitor. The eel gill TA-receptor complex sedimented on a linear (10–30%) sucrose gradient with a single peak at 7.0 S or 3.5 S, in hypotonic or hypertonic (0.4 M KCl) gradients, respectively. The eel ligand-receptor complex did not bind, following heat activation, to DNA-cellulose or phosphocellulose, though it bound to DEAE-cellulose. In this respect, it behaved similarly to the eel intestinal mucosal TA-receptor complex, described previously. The initiation of dissociation of the eel receptor-[³H]TA complex with excess TA yielded pseudo-first-order dissociation kinetics (k_?1 at 0°C: 2.39 × 10^?5 s^?1), while the association kinetics of the receptor with the ligand was of second order (k₊₁: 2.51 × 10⁴M^?1 s^?1). Sepharose column chromatography indicated a molecular weight of 334,690 Da. Calculation of the Stokes radius gave a value of 84.5 Å and the frictional ratio, calculated from the molecular weight, was 1.84. From these data it was concluded that the gills of these two euryhaline teleosts contain tetrapod-type glucocorticoid receptors. These studies are the first to demonstrate these steroid recognition molecules in fish gill. The presence of receptors in the fish gill tissue are in agreement with the physiological action of corticosteroids in allowing adaptation of the animals to habitats of different salinity.     

Immunohistochemical detection of estrogen receptors in fish scales

Calcium mobilization from internal stores, such as scales, induced by 17β-estradiol during sexual maturation in salmonids is well documented. This calcium mobilization from scales is proposed to be mediated by the estrogen receptor (ER). However, the ER subtypes involved and signaling mechanisms responsible for this effect remain to be fully characterized. In the present study, we have localized ERα, ERβa and ERβb proteins in juvenile and adult sea bream (Sparus auratus) and Mozambique tilapia (Oreochromis mossambicus) scales by immunohistochemistry with sea bream ER subtype specific antibodies. The three ERs were detected in isolated or small groups of round cells, in the basal layer of the scales of both juvenile and adult fish and the localization and signal intensity varied with the species and age of the animals. The ERs may be co-localized in cells of the scale posterior region that expressed tartrate-resistant acid phosphatase (TRAP), a marker for osteoclasts. These results suggest that the calcium mobilizing action of 17β-estradiol on fish scales is via its direct action on ERs localized in osteoclasts.     

Regulation of somatostatins and their receptors in fish

The multifunctional nature of the somatostatin (SS) family of peptides results from a multifaceted signaling system consisting of many forms of SS peptides that bind to a variety of receptor (SSTR) subtypes. Research in fish has contributed important information about the components, function, evolution, and regulation of this system. Somatostatins or mRNAs encoding SSs have been isolated from over 20 species of fish. Peptides and deduced peptides differ in their amino acid chain length and/or composition, and most species of fish possess more than one form of SS. The structural heterogeneity of SSs results from differential processing of the hormone precursor, preprosomatostatin (PPSS), and from the existence of multiple genes that give rise to multiple PPSSs. The PPSS genes appear to have arisen through a series of gene duplication events over the course of vertebrate evolution. The numerous PPSSs of fish are differentially expressed, both in terms of the distribution among tissues and in terms of the relative abundance within a tissue. Accumulated evidence suggests that nutritional state, season/stage of sexual maturation, and many hormones [insulin (INS), glucagon, growth hormone (GH), insulin-like growth factor-I (IGF-I), and 17beta-estradiol (E2)] regulate the synthesis and release of particular SSs. Fish and mammals possess multiple SSTRs; four different SSTRs have been described in fish and several of these occur as isoforms. SSTRs are also wide spread and are differentially expressed, both in terms of distribution of tissues as well as in terms of relative abundance within tissues. The pattern of distribution of SSTRs may underlie tissue-specific responses of SSs. The synthesis of SSTR mRNA and SS-binding capacity are regulated by nutritional state and numerous hormones (INS, GH, IGF-I, and E2). Accumulated evidence suggests the possibility of both tissue- and subtype-specific mechanisms of regulation. In many instances, there appears to be coordinate regulation of PPSS and of SSTR; such regulation may prove important for many processes, including nutrient homeostasis and growth control.     

Epidermal growth factor decreases the concentration of thyrotropin-releasing hormone (TRH) receptors and TRH responses in pituitary GH4C1 cells.

Treatment of pituitary GH4C1 cells with epidermal growth factor (EGF) caused up to a 60% reduction in the amount of [3H]MeTRH bound to specific TRH receptors. The effects of EGF were first detectable after a 2-h incubation and maximal by 24-72 h. EGF elicited a half-maximal response at 0.03 nM. Equilibrium binding analysis was performed on intact cells that had been incubated with or without 10 nM EGF for 96 h. EGF decreased the apparent number of TRH receptors (maximum binding = 0.36 vs. 0.58 pmol/mg protein for EGF-treated and control cells, respectively) without altering the apparent affinity (dissociation constant = 6.4 vs. 7.4 nM). The effects of EGF on TRH receptors were reversible. When EGF was removed from the medium, TRH receptors returned to control levels within 48 h. To assess whether the reduction of TRH receptors was functionally important, the ability of TRH to stimulate phospholipid turnover was measured in cells with a normal complement of TRH receptors and in cells that had been treated with EGF for 72 h to reduce TRH receptor density. EGF significantly blunted the ability of TRH to stimulate release of inositol phosphates from metabolically labeled cells. TRH increased inositol monophosphate accumulation 6.3-fold in control cultures and 2.0-fold in EGF-treated cells. These data show that EGF regulates the concentration of TRH receptors on pituitary GH4C1 cells and the responsiveness of the cells to TRH.     

Perspectives on fish gonadotropins and their receptors

Teleosts lack a hypophyseal portal system and hence neurohormones are carried by nerve fibers from the preoptic region to the pituitary. The various cell types in the teleost pituitary are organized in discrete domains. Fish possess two gonadotropins (GtH) similar to FSH and LH in other vertebrates; they are heterodimeric hormones that consist of a common α subunit non-covalently associated with a hormone-specific β subunit. In recent years the availability of molecular cloning techniques allowed the isolation of the genes coding for the GtH subunits in 56 fish species representing at least 14 teleost orders.Advanced molecular engineering provides the technology to produce recombinant GtHs from isolated cDNAs. Various expression systems have been used for the production of recombinant proteins. Recombinant fish GtHs were produced for carp, seabream, channel and African catfish, goldfish, eel, tilapia, zebrafish, Manchurian trout and Orange-spotted grouper.The hypothalamus in fishes exerts its regulation on the release of the GtHs via several neurohormones such as GnRH, dopamine, GABA, PACAP, IGF-I, norepinephrine, NPY, kisspeptin, leptin and ghrelin. In addition, gonadal steroids and peptides exert their effects on the gonadotropins either directly or via the hypothalamus. All these are discussed in detail in this review.In mammals, the biological activities of FSH and LH are directed to different gonadal target cells through the cell-specific expression of the FSH receptor (FSHR) and LH receptor (LHR), respectively, and the interaction between each gonadotropin-receptor couple is highly selective. In contrast, the bioactivity of fish gonadotropins seems to be less specific as a result of promiscuous hormone–receptor interactions, while FSHR expression in Leydig cells explains the strong steroidogenic activity of FSH in certain fish species.     

Role of the hypothalamic TRH in the regulation of its own receptors in rat anterior pituitaries

We explored whether endogenous TRH in the hypothalamus produced a potential change in its receptors in the rat anterior pituitary. Thyroidectomy caused a progressive increase in the pituitary TRH binding with a concomitant increase in the blood TSH level. Injection of T4 prevented the increase in the pituitary TRH binding and the blood TSH level after thyroidectomy. The hypothalamic deafferentation and electrolytic lesions in the hypothalamic paraventricular nuclei led to a significant reduction in both the content of TRH in the hypothalamic median eminence and the blood TSH level, but they affected neither the number nor affinity constant of the pituitary TRH receptors in thyroidectomized rats. The present data provide evidence that TRH receptors in the anterior pituitary are profoundly regulated by thyroid hormones, but not significantly by TRH in the hypothalamus in the rat.     

TRH analog administration increases endogenous TRH levels in the central nervous system

Experiments were carried out to increase the endogenous levels of TRH in brain and spinal cord. After 7 days of continuous oral ingestion of TRH analogs (D-pGlu-Leu-ProNH2, pGlu-Leu-Pro, pGlu-Pro-ProNH2, or pGlu-Leu-pyrrolidide), spinal cord and brain levels of TRH were elevated, whereas the content of TRH metabolite, cyclo(His-Pro), was decreased. In addition, the TRH analogs inhibited in vitro metabolism of [3H-Pro]-TRH by brain and spinal cord extracts. Our data show that certain TRH analogs can elevate tissue levels of TRH by inhibiting its metabolism. Furthermore, because these analogs do not affect interaction between TRH and its receptor, the elevated TRH can effectively enhance TRH-related bioactivity.     

Relationships between aromatase and estrogen receptors in the brain of teleost fish

Teleost fish are known for exhibiting a high aromatase activity mainly due to the expression of the cyp19b gene, encoding aromatase B (AroB). Recent studies based on both in situ hybridization and immunohistochemistry have demonstrated in three different species that this activity is restricted to radial glial cells. In agreement with measurements of aromatase activity, such aromatase-expressing cells are more abundant in the telencephalon, preoptic area, and mediobasal hypothalamus, although positive cells are also found in the midbrain and hindbrain. Comparative distribution of AroB and estrogen receptor (ERalpha, ERbeta1, and ERbeta2) expression indicates that the preoptic region and hypothalamus are major target for locally produced estradiol (E2) which is likely involved in controlling expression of genes implicated in neuroendocrine regulations. However, AroB and ER have never been reported to be co-expressed in the same cells which is intriguing given that, at least in some species, AroB is strongly up-regulated by E2 itself in agreement with the presence of an estrogen-responsive element (ERE) in the proximal promoter of the cyp19b gene. In vivo data in zebrafish have shown that E2 up-regulates AroB only in radial glial cells. This is in agreement with in vitro transfection experiments indicating that this ERE is functional, but not sufficient, as the E2 regulation of AroB only occurs in glial cell contexts, suggesting a cooperation between ER and so far unidentified glial-specific factors. These data also suggest that radial glial cells may express low amounts of ER that escaped detection until now. The expression of AroB in radial cells, well known for their roles in neurogenesis and now considered as progenitor cells, suggests that local E2 production within these cells could influence the well-documented capacity of the brain of teleosts to grow during adulthood.     

Neurokinin Bs and neurokinin B receptors in zebrafish-potential role in controlling fish reproduction

The endocrine regulation of vertebrate reproduction is achieved by the coordinated actions of several peptide neurohormones, tachykinin among them. To study the evolutionary conservation and physiological functions of neurokinin B (NKB), we identified tachykinin (tac) and tac receptor (NKBR) genes from many fish species, and cloned two cDNA forms from zebrafish. Phylogenetic analysis showed that piscine Tac3s and mammalian neurokinin genes arise from one lineage. High identity was found among different fish species in the region encoding the NKB; all shared the common C-terminal sequence. Although the piscine Tac3 gene encodes for two putative tachykinin peptides, the mammalian ortholog encodes for only one. The second fish putative peptide, referred to as neurokinin F (NKF), is unique and found to be conserved among the fish species when tested in silico. tac3a was expressed asymmetrically in the habenula of embryos, whereas in adults zebrafish tac3a-expressing neurons were localized in specific brain nuclei that are known to be involved in reproduction. Zebrafish tac3a mRNA levels gradually increased during the first few weeks of life and peaked at pubescence. Estrogen treatment of prepubertal fish elicited increases in tac3a, kiss1, kiss2, and kiss1ra expression. The synthetic zebrafish peptides (NKBa, NKBb, and NKF) activated Tac3 receptors via both PKC/Ca(2+) and PKA/cAMP signal-transduction pathways in vitro. Moreover, a single intraperitoneal injection of NKBa and NKF significantly increased leuteinizing hormone levels in mature female zebrafish. These results suggest that the NKB/NKBR system may participate in neuroendocrine control of fish reproduction.     

TRH stimulation of in vivo GH release in the domestic fowl. Influence of TRH metabolites and effect of age on in vitro degradation of TRH

Thyrotropin-releasing hormone (TRH) stimulated in vivo growth hormone (GH) release in conscious and anesthetized young domestic fowl. The administration of the presumed metabolites of TRH, deamido-TRH (TRH-OH) and histidyl-proline diketopiperazine (HPD), was followed by small but significant (p less than 0.05) increases in the plasma concentrations of GH in both conscious and anesthetized chicks. However, the ability of TRH-OH or HPD to stimulate GH secretion was less than that observed with a 100-fold lower dose of TRH. The administration of either TRH-OH or HPD with TRH increased the GH response over that observed with TRH alone. The ability of chicken plasma to degrade exogenous TRH in vitro was determined by measuring immunoreactive TRH (IR-TRH) content and by assessing the ability of the incubated samples to increase the plasma concentration of GH when administered to young fowl. The in vitro half-life of TRH was estimated to be 9.8 (by immunoassay) and 9.6 (by a biological index) min for plasma from adult male chickens and 23.9 (by immunoassay) and 20.2 (by biological index) min for plasma from 6-week-old chicks. This difference in degradation may account, at least in part, for the observed age-related decrease in the plasma concentration of GH in birds and for the diminished GH responsiveness of adult birds to exogenous TRH.