Degradation of gonadotropin-releasing hormones in the gilthead seabream, Sparus aurata. II. Cleavage of native salmon GnRH, mammalian LHRH, and their analogs in the pituitary, kidney, and liver

The pattern and kinetics of degradation of native salmon gonadotropin-releasing hormone (sGnRH), mammalian luteinizing hormone-releasing hormone (LHRH), and some of their analogs by cytosolic enzymes of pituitary, kidney, and liver were studied in the gilthead seabream, Sparus aurata. The native peptides sGnRH and LHRH are rapidly degraded by all three tissues, LHRH being degraded faster than sGnRH. The kinetics of production of the peptide fragments suggest that initial cleavage of sGnRH and LHRH in the three studied tissues occurs at the 5-6 and 9-10 bonds. This indicates the initial activity of a Tyr5-Gly6 endopeptidase and a Pro9-Gly10NH2 peptidase or postproline cleaving enzyme. Secondary degradation of the main initial fragments (1-5, 6-10, and 1-9) is more intensive in the kidney than in the pituitary or liver. Substitution of the position 6 amino acid glycine by a dextrorotatory (D) amino acid such as in the D-Trp6-LHRH renders the 5-6 bond resistant to cleavage. However, whereas [D-Trp6]-LHRH is intensively cleaved at the Pro9-Gly10NH2 bond by the pituitary, its cleavage at this site by the kidney and liver is slow. This suggests a low activity of the Pro9-Gly10NH2 peptidase in the kidney and liver as compared to the pituitary. When, in addition to the position 6 substitution, the carboxy terminus Pro9-Gly10NH2 is modified to Pro9NET, such as in the [D-Ala6-Pro9NET]-LHRH and the [D-Arg6-Pro9NET]-sGnRH, the 9-10 cleavage site is also blocked, resulting in GnRH analogs highly resistant to degradation. The relationships between susceptibility of the different forms of GnRH to enzymatic degradation by the pituitary, kidney, and liver and their relative biological activities in S. aurata are discussed. We conclude that increased resistance of GnRH analogs to enzymatic degradation contributes to their superactivity.     

Degradation of gonadotropin-releasing hormones in the gilthead seabream, Sparus aurata. I. Cleavage of native salmon GnRH and mammalian LHRH in the pituitary

The pattern and kinetics of degradation of native salmon gonadotropin-releasing hormone (sGnRH) and mammalian leuteinizing hormone-releasing hormone (LHRH) by pituitary bound enzymes were studied in the gilthead seabream, Sparus aurata. sGnRH and LHRH were incubated for different periods of time with membrane or cytosolic fractions of pituitary homogenates. At the end of the incubation, the degradation mixture was fractionated on reverse-phase high-pressure liquid chromatography. The degradation products were identified by comparing their retention times to those of synthesized GnRH fragments and by analyzing their amino acid composition. The main GnRH degradative activity resides in the cytosolic fraction of the pituitary homogenate. Both sGnRH and LHRH are rapidly degraded by pituitary cytosol, with 78.3 and 87.7% of the peptides, respectively, cleaved after 3 hr of incubation. Maximal degradation of sGnRH occurred at a pH range of 7 to 8. The main initial products of degradation of sGnRH and LHRH are the 1-5, 6-10, and 1-9 fragments. This suggests the involvement of two site-specific peptidases, a Tyr5-Gly6 endopeptidase and a Pro9-Gly10NH2 peptidase or postproline cleaving enzyme. While the 1-6 and 1-9 fragments undergo rapid secondary degradation, the 1-5 is relatively stable. Competition experiments suggest that the endopeptidase cleaving the sGnRH at the Tyr5-Gly6 bond is not specific to the neuropeptide and is probably a general proteolitic enzyme. However, the cleavage at the 9-10 bond has a high degree of specificity to the Pro9-Gly10NH2 sequence found in sGnRH. The two proposed pituitary peptidases of S. aurata have some characteristics similar to those of rat hypophyseal and hypothalamic GnRH cleaving enzymes. No differences are found in hypophyseal GnRH degradative activity between females with occytes undergoing previtellogenesis or advanced stages of vitellogenesis.     

Use of a pituitary cell dispersion method and primary culture system for the studies of gonadotropin-releasing hormone action in the goldfish, Carassius auratus. I. Initial morphological, static, and cell column perifusion studies

Two cell dispersion methods for excised goldfish pituitary glands were tested, and a cultured dispersed cell system based on trypsin enzymatic tissue digestion was developed and characterized. Controlled trypsin/DNase treatment of goldfish pituitary gland yielded dispersed cells of high viability (trypsin blue exclusion test) that responded to gonadotropin (GTH)-releasing hormone (GnRH) challenges with GTH secretion in a time- and dose-dependent manner following overnight culture. Electron microscopy revealed that cell preparations produced by the trypsin dispersion were free of cell debris and nerve terminals. The dispersed pituitary cells also retained distinct morphological and immunological identities. Under static incubation conditions, 2-hr treatments with 0.1 nM to 1 microM [Trp7,Leu8]-GnRH (sGnRH) and [D-Arg6,Pro9-N-ethylamide]-sGnRH (sGnRHa) stimulated GTH release with similar efficacy, but with ED50S of 1.92 +/- 0.48 and 0.19 +/- 0.08 nM, respectively. [His5,Trp7,Tyr8]-GnRH (cGnRH-II) stimulated GTH release in a nonsigmoidal, but dose-dependent manner, and with a higher efficacy than sGnRH. In contrast, sGnRH, sGnRHa, and cGnRH-II were equipotent in inducing growth hormone (GH) secretion in static culture studies and with ED50S of 0.29 +/- 0.13, 0.18 +/- 0.11, and 0.19 +/- 0.17 nM, respectively. When trypsin/DNase-dispersed cells cultured overnight with cytodex beads were tested in a cell column perifusion system, dose-related increase in GTH secretion, as well as GH release, were also observed with 0.5 to 50 nM sGnRH. These results suggest that trypsin-dispersed goldfish pituitary cells can be used effectively to study the actions of GnRH on teleost pituitary either in short-term static incubation or column perifusion studies. Differences in the GTH and GH responses to the two native GnRH forms, sGnRH and cGnRH-II, are also indicated.     

Desensitization to native molecular forms of gonadotropin-releasing hormone in the goldfish pituitary: dependence on pulse frequency and concentration.

Homologous desensitization of gonadotropin-releasing hormone (GnRH) was investigated using goldfish pituitary fragments in vitro. The two native GnRH peptides, sGnRH [( Trp7, Leu8]-GnRH) and cGnRH-II [( His5, Trp7, Tyr8]-GnRH) were administered either continuously or in pulsatile fashion at different frequencies and concentrations. Continuous treatment (60 min) with either sGnRH or cGnRH-II at 10(-7), 10(-8), and 10(-9) M resulted in desensitization of goldfish pituitary in a biphasic fashion, characterized by an initial rapid peak of GTH release (phase 1), followed by a lower sustained release of GTH remaining at a stable concentration above the basal level (phase 2). Pititary fragments were then washed for 60 min and further treated continuously (60 min) with the same concentrations of sGnRH or cGnRH-II (second treatment). Total sGnRH- or cGnRH-II-induced GTH release during the second treatment period was significantly lower than that observed during the initial treatment period, depending upon the concentration of the peptides. The second phase of GTH release was more pronounced at lower concentrations compared to that observed following 10(-7) M treatment, especially for sGnRH. Pulsatile treatment with either sGnRH or cGnRH-II (2-min pulses of 10(-7), 10(-8), and 10(-9) M given every 20 min) resulted in significant desensitization of the pituitary GTH release. Reduction of pulse frequency to 2 min treatment every 60 min resulted in a lower degree of desensitization; little or no desensitization was observed following treatment with 10(-8) and 10(-9) M cGnRH-II or 10(-9) M sGnRH. A further reduction in frequency to 2-min pulses of sGnRH or cGnRH-II (10(-7) or 10(-8) M) given every 90 min did not result in desensitization of the pituitary GTH release. In summary, the present study demonstrates that GnRH-induced desensitization is dependent on both pulse frequency and concentration in the goldfish pituitary. These findings support the hypothesis that pulsatile secretion of the native GnRH peptides may be essential for maintenance of normal pituitary GTH release in goldfish.     

Characterization of gonadotropin-releasing hormone binding sites in the pituitary of the three-spined stickleback, Gasterosteus aculeatus

Binding sites for gonadotropin-releasing hormone (GnRH) in stickleback pituitary homogenates were characterized using an iodinated, superactive analog of salmon GnRH (sGnRH), D-Arg6-Pro9-sGnRH-NEt (sGnRHa). Binding of 125I-sGnRHa reached equilibrium after 60 min incubation at 4 degrees and was a function of tissue concentration. The specificity of 125I-sGnRHa binding was demonstrated by displacement with sGnRHa, sGnRH, and Buserelin [D-Ser(t-Bu)6-Pro9-GnRH-NEt]. Both Scatchard analyses of saturation data and displacement curves revealed a single class of high-affinity binding sites (Ka = 0.71 +/- 0.03 X 10(9) M-1, Bmax = 1087 +/- 165 fmol/mg protein).     

Participation of arachidonic acid metabolism in gonadotropin-releasing hormone stimulation of goldfish gonadotropin release

Two intraperitoneal injections of a mammalian gonadotropin-releasing hormone (GnRH) analog, [D-Ala6, Pro9-N-ethylamide]-GnRH (mGnRHa; 0.1 micrograms/g), at 12-hr intervals increased serum gonadotropin (GTH) levels in sexually mature and sexually regressed female goldfish 2 and 6 hr after the second injection. This serum GTH response was decreased by the coinjection of a lipoxygenase enzyme inhibitor, nordihydroguaiaretic acid (NDGA: 0.1 to 10 micrograms/g) at the time of the second mGnRHa application. In static cultures of dispersed goldfish pituitary cells, 1-100 microM arachidonic acid (AA) and 0.1-1000 nM [Trp7, Leu8]-GnRH (salmon GnRH, sGnRH) and [D-Arg6, Pro9-N-ethylamide]-sGnRH (sGnRHa) caused dose-dependent increases in GTH release. Additions of 1-40 microM NDGA reduced the sGnRH-stimulated GTH release in a dose-dependent manner, and completely inhibited the GTH response to increasing concentrations of AA. NDGA 40 microM also decreased the elevated GTH levels induced by sGnRHa treatment. Exposure to 10 microM 5,8,11,14-eicosatetraynoic acid, an inhibitor with mixed action on lipoxygenase and cyclooxygenase enzymes, reduced the dose-dependent GTH response to sGnRH and AA. In contrast, coincubation with another cyclooxygenase blocker, indomethacin, at 10 microM, did not alter AA and sGnRH-induced GTH release. These results provide in vivo and in vitro evidence for the participation of AA metabolism in mediating GnRH-stimulated GTH release in the goldfish. The importance of AA metabolism through the lipoxygenase pathway is also indicated.     

Evidence that gonadotropin-releasing hormone also functions as a growth hormone-releasing factor in the goldfish

The present study examined the influence of GnRH on the in vivo and in vitro secretion of GH in the goldfish (Carassius auratus). Intraperitoneal injection of several GnRH peptides, including a form native to goldfish, salmon GnRH (sGnRH), elevated circulating GH levels in female goldfish. An analog of mammalian GnRH (mGnRH), [D-Ala6,Pro9-NEt] mGnRH (mGnRH-A), at a dosage of 0.1 microgram/g BW increased serum GH levels for up to 48 h after a single ip injection. Goldfish receiving a series of injections of this dose of mGnRH-A also displayed an increased rate of body growth, indicating that the mGnRH-A-induced increase in the circulating GH level was sufficient to accelerate body growth. In vitro experiments using perifused pituitary fragments found that sGnRH stimulated the secretion of GH from the goldfish pituitary in a potent, dose-dependent, and reversible manner. The time course of response and half-maximally effective dose of sGnRH were very similar for both GH and gonadotropin (GTH) secretion in vitro, suggesting that the mechanism(s) mediating the stimulatory actions of GnRH in the goldfish may be similar for both GH and GTH secretion. However, GnRH-induced GH and GTH secretion from the goldfish pituitary can occur independently of each other, as demonstrated by the finding that somatostatin inhibited the GnRH stimulation of GH secretion in vitro, without influencing the GTH response, whereas the dopamine agonist apomorphine inhibited GnRH-induced GTH secretion in vitro, without influencing the GH response. Furthermore, the dopamine antagonist pimozide did not influence serum GH levels, although pimozide potentiated the stimulatory effect of GnRH on GTH secretion in vivo by blocking the endogenous GTH release inhibitory action of dopamine. Results of the present study suggest that the secretion of GH and GTH in the goldfish are regulated, at least in part, through a common releasing factor, GnRH, whereas somatostatin and dopamine appear to act independently as GH and GTH release inhibitory factors, respectively.     

Use of a pituitary cell dispersion method and primary culture system for the studies of gonadotropin-releasing hormone action in the goldfish, Carassius auratus. II. Extracellular calcium dependence and dopaminergic inhibition of gonadotropin responses

Primary static cultures of dispersed goldfish pituitary cells obtained by controlled trypsinization released gonadotropin (GTH) in response to 2-hr stimulations of 0.1 nM to 1 microM [Trp7,Leu8]-gonadotropin-releasing hormone (sGnRH), [D-Arg6,Pro9-N-ethylamide]-sGnRH (sGnRHa), and [His5,Trp7,Tyr8]-GnRH (cGnRH-II) in a dose-dependent manner. Coincubation with 10 to 1000 nM of a dopamine agonist, apomorphine, dose dependently reduced the GTH response to increasing concentrations of sGnRH. Apomorphine at 1 microM completely abolished the dose-dependent GTH response to sGnRHa and cGnRH-II, but only partially inhibited the GTH-releasing action of high concentrations of sGnRH. Addition of calcium ionophores, 1 to 100 microM A23187 and 10 to 100 microM ionomycin, significantly increased GTH release. The ED50S of the GTH response to A23187 and ionomycin were 0.88 +/- 0.15 and 13.67 +/- 2.76 microM, respectively. Incubation with Ca2(+)-deficient media (media prepared without the addition of Ca2+ salts) did not significantly affect basal GTH release, but severely decreased the hormone response to increasing concentrations of sGnRH, A23187, and ionomycin. These results confirm the direct inhibitory dopaminergic influence on GTH release in goldfish and further suggest that extracellular Ca2+ plays a role in mediating GnRH action on gonadotropes in fish.     

A study of LH-RH receptors in the pituitary gland of the winter flounder (Pseudopleuronectes americanus Walbaum)

A study using an iodinated [D-Ser(tBu)6,Pro9-NHEt]LH-RH (Buserelin), demonstrated the presence of a single class of high-affinity (KD = 2.90 nM), high-capacity LH-RH binding sites in pituitaries obtained from sexually mature male and female winter flounder. Displacement curves for unlabeled Buserelin and other preparations of mammalian and fish LH-RH, but a lack of competition for structurally unrelated peptide hormones, indicated the hormone specific nature of the fish pituitary LH-RH receptor preparation. Compared with native mammalian LH-RH and salmon LH-RH, Buserelin and an analog of salmon LH-RH, [D-Arg6,Trp7,Leu8,Pro9-NHEt]LH-RH, had significantly higher binding affinities for the flounder pituitary receptor correlating with results of previous studies demonstrating the superagonist biological activity of LH-RH analogs in trout and goldfish.     

Differences in salmon GnRH and chicken GnRH-II contents in discrete brain areas of male and female rainbow trout according to age and stage of maturity

We have developed sensitive and specific radioimmunoassays (RIA) for salmon gonadotropin-releasing hormone (sGnRH) and chicken GnRH-II (cGnRH-II). Synthetic sGnRH and cGnRH-II(2-10) were conjugated to bovine serum albumin and injected into rabbits to raise specific antisera. The antiserum against sGnRH showed cross-reactivities of 1.58 and 0.08% for cGnRH-II and lamprey GnRH, respectively. The antiserum against cGnRH-II showed cross-reactivities of 0.05 and 0.01% for sGnRH and lamprey GnRH, respectively. Both antisera were observed not to cross-react with mammalian GnRH and cGnRH-I or other peptide hormones. Synthetic sGnRH and cGnRH-II were iodinated using the chloramine-T method. The iodinated GnRH was purified by HPLC using a reverse-phase C18 column. The RIA system was developed as a double antibody method. Brain extracts of rainbow trout showed displacement curves which were parallel to the sGnRH and cGnRH-II standards in each RIA. HPLC analysis followed by RIA has revealed that rainbow trout brain contains two types of GnRH: sGnRH and cGnRH-II. Total sGnRH content in the brain was about three-fold higher than that of cGnRH-II. In the olfactory bulbs, telencephalon, optic tectum-thalamus, hypothalamus, and pituitary, sGnRH content (per region) was higher than cGnRH-II content, whereas cerebellum and medulla oblongata contained much more cGnRH-II than sGnRH. sGnRH content in the optic tectum-thalamus and pituitary was the highest in 1-year-old immature fish and 3-year-old mature fish, respectively. Medulla oblongata showed the highest cGnRH-II content in all groups. sGnRH concentrations (per milligram of protein) were high in the pituitary and intermediate in the olfactory bulbs, hypothalamus, and telencephalon. In all groups, the cGnRH-II concentration was high in the medulla oblongata, whereas the concentration in the olfactory bulbs and pituitary gland was below the detectable limit in most individuals.     

Use of pituitary cells in primary culture to study the regulation of gonadotropin hormone (GtH) secretion in rainbow trout: setting up and validating the system as assessed by its responsiveness to mammalian and salmon gonadotropin releasing hormone

To study the regulation of gonadotropin secretion in rainbow trout in vitro, a method for preparing primary cultures of dispersed pituitary cells is described. Cells were dispersed by collagenase 0.1% in Hank's saline solution for 20 hr at 12 degrees and a high yield of viable cells was obtained. Attempts to improve cell functioning were made by varying culture conditions (density of cells initially plated, age of the culture). Cell functioning was assessed by their ability to respond to increasing doses of mammalian and salmon GnRH. Pituitaries were collected from spermiating males whose pituitaries are known to be sensitive to mammalian GnRH in vivo. Using 96-well plates, optimal conditions for good biological activity, are initial plating with 6.2 X 10(4) cells, incubation with GnRH for 24 hr on the third day after plating. In these conditions mammalian analog and salmon GnRH induced an increase in GtH release for doses ranging from 10(-9) to 10(-6) M. The GtH released during the GnRH incubation period does not decrease the sensitivity of the system since addition of 20 ng of GtH at the beginning of incubation does not modify the response profile.     

Cultured pituitary cell GtH response to GnRH at different stages of rainbow trout spermatogenesis and influence of steroid hormones

Using primary cultures of whole dispersed pituitary cells collected from rainbow trout at different stages of spermatogenesis, basal and GnRH-induced GtH release and cell GtH content were studied in control and steroid-pretreated cultures. Steroid pretreatments were performed for 3 days with 11-ketotestosterone (11KT) and 17 alpha-hydroxy,20 beta-dihydroprogesterone (17 alpha 20 beta p) at levels corresponding to those circulating at the time of spermiation (50 and 20 ng/ml, respectively). In control cultures, basal GtH release and cell GtH content increased with the stage of spermatogenesis in a characteristic pattern as predicted from in vivo results concerning plasma and pituitary GtH contents. The pituitary response to salmon GnRH (sGnRH) also varied as indicated by the decrease in the minimal effective dose of GnRH able to induce a significant GtH release with the advancement of spermatogenesis: 10(-7) M at the spermatocyte stage, 10(-9) M at prespermiation and spermiation. Steroid pretreatments were shown to have a direct effect on pituitary gonadotrophs and particularly on pituitary response to sGnRH, depending on the stage at which they are applied. At the beginning of spermatogenesis both of them induced an increase in GtH release and at prespermiation they have a slight negative effect, significant only with 17 alpha 20 beta P. At spermiation they have no effect except for 17 alpha 20 beta P which increased the response to 10(-8) M of sGnRH. Results are discussed in relation to hormonel changes (gonadotropin and steroid) observed by different authors during the sexual cycle.     

Relative activity, plasma elimination and tissue degradation of synthetic luteinizing hormone releasing hormone and certain of its analogues.

The abilities of three nonapeptide analogues of synthetic luteinizing hormone releasing hormone (LH-RH) to release luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in anoestrous and cyclic ewes were examined, as were their elimination from the plasma in vivo and degradation by extracts of the hypothalamus, anterior pituitary gland, lung, kidney, liver and plasma in vitro. In all cases, comparisons were made with synthetic LH-RH. When injected i.v. into mature ewes as a single dose, the potencies of the analogues were graded and Des Gly-NH2(10) LH-RH ethylamide was found to be the least potent. It was not possible to demonstrate any significant increase in the potency of this analogue over LH-RH, although a trend was apparent with each parameter examined. [D-Ser(But)6] Des Gly-NH2(10) LH-RH ethylamide had the greatest potency. There were no differences between the responses of anoestrous ewes and those of ewes treated on day 10 of the oestrous cycle. None of the analogues had a rate of elimination from the plasma different from that of LH-RH during either the first or the second components of the biphasic disappearance curve. The incubation of LH-RH with tissue extracts showed that extracts of the hypothalamus and anterior pituitary gland degraded LH-RH to a similar extent. Both the hypothalamic and anterior pituitary gland extracts degraded more LH-RH than did lung extract, which in turn destroyed more LH-RH than did extracts of kidney or liver tissue. The degradative abilities of kidney and liver extracts did not differ from each other. Plasma failed to degrade LH-RH or the analogues. Although LH-RH was rapidly destroyed by hypothalamic extract in vitro, of the analogues, only Des Gly-NH2(10) LH-RH ethylamide was degraded. The anterior pituitary gland and kidney extracts failed to degrade [D-Ser6] Des Gly-NH2(10) LH-RH ethylamide and [D-Ser(But)6] Des Gly-NH2(10) LH-RH ethylamide as rapidly as LH-RH. Extracts of liver and lung were incapable of catabolizing any of the analogues. There was an inverse correlation between the LH- and FSH-releasing potency of an analogue and its rate of degradation by anterior pituitary gland extract. The slower rates of catabolism of certain analogues of LH-RH by the anterior pituitary gland may explain their increased LH- and FSH-releasing potency.     

Biphasic effects of estrogen on gonadotropin-releasing hormone-induced luteinizing hormone release in monolayer cultures of rat and monkey pituitary cells

The negative feedback effect of estrogen on LH secretion has been difficult to demonstrate in monolayer cultures of rat pituitary cells. The purpose of the present study was to establish the experimental conditions required for manifestation of this response and, in the process, to develop models for investigating the actions of steroids on pituitary cells. Both dynamic and static incubation systems were used. For perifusion experiments, trypsin-dispersed pituitary cells from rats at random stages of the estrous cycle were attached to glass coverslips with poly-L-lysine, incubated for 48 h, and then mounted in Sykes-Moore chambers. In each of these experiments, two chambers were perifused concurrently: one with medium containing 1.8 X 10(-10) M 17 beta-estradiol and the other with medium alone. GnRH (4.2 X 10(-9) M) was coinfused for 5 min out of every hour, and samples of perifusate were collected as 5-min fractions for assay of LH. Estrogen treatment significantly (P less than 0.01) suppressed LH release in response to the first five GnRH pulses compared to the control value. The inhibition was most pronounced early in the perifusion, but had disappeared by 6 h. These results demonstrate that estradiol exerts a potent but transient inhibition of GnRH-induced LH release in monolayer cultures of rat pituitary cells. In a subsequent set of experiments, we modified a static incubation system to assess sequentially the biphasic effects of estrogen on LH release by the same group of cells. Cultures of rat pituitary cells that had been established 42 h previously were treated simultaneously for 3 h with 17 beta-estradiol (3.7 X 10(-10) M) and various concentrations of GnRH (5 X 10(-10) to 1 X 10(-7) M) to measure the inhibitory effects of the steroid on LH secretion. This experiment was repeated on the same cells after 27 h of steroid exposure to estimate the facilitory actions of estrogen on LH release. The negative feedback of estrogen was demonstrable in static cultures of rat pituitaries provided that the period of estrogen exposure and duration of incubation were brief. Moreover, the results indicate that the same groups of cells can be used on consecutive days to investigate the inhibitory and stimulatory effects of estrogen on LH secretion. Experiments with cultures of monkey pituitary cells yielded similar results. Taken together, these findings indicate that cultured pituitary cells are responsive to the biphasic actions of estradiol and demonstrate the utility of two model systems for investigating these phenomena.     

Studies of the biological activity of LHRH analogs in the rainbow trout, landlocked salmon, and the winter flounder

Studies of gonadotropic hormone (GtH) release bioactivity by mammalian and submammalian varieties of LHRH and LHRH analog were primarily conducted in vivo in testosterone-primed yearling (TPY) rainbow trout, a convenient test animal for LHRH bioassays in fish. Validation of these results, using sexually mature fish, was accomplished by examining LHRH agonist activities on release of GtH in vivo in spermiating landlocked salmon and by studying LHRH peptide hormone binding affinities using a flounder pituitary LHRH radioreceptor assay. Our surveys of LHRH analog bioactivity in vivo in TPY trout and salmon demonstrated that all types of fish, bird, and mammalian LHRH agonists possess superactive properties on the fish pituitary. The most active group of LHRH analogs, based upon both LHRH receptor binding affinity and in vivo release of gonadotropin, was judged to include [D-Nal(2)6,Pro9-NHEt]LHRH, [D-Nal(2)6-AzaGly10]LHRH, [D-Ala6,Pro9-NHEt]-LHRH, and the fish LHRH analogs, [D-Arg6,Trp7,Leu8,Pro9-NHEt]LHRH, [D-hArg(Et2(6),Trp7,Leu8,Pro9-NHEt]LHRH.     

Highly potent analogues of luteinizing hormone-releasing hormone containing D-phenylalanine nitrogen mustard in position 6.

The nitrogen mustard derivatives of 4-phenylbutyric acid and L-phenylalanine, called chlorambucil (Chl) and melphalan (Mel), respectively, have been incorporated into several peptide hormones, including luteinizing hormone-releasing hormone (LH-RH). The alkylating analogues of LH-RH were prepared by linking Chl, as an N-acyl moiety, to the complete amino acid sequence of agonistic and antagonistic analogues. These compounds, in particular the antagonistic analogues, showed much lower potency than their congeners carrying other acyl groups. To obtain highly potent alkylating analogues of LH-RH, the D enantiomer of Mel was incorporated into position 6 of the native hormone and some of its antagonistic analogues. Of the peptides prepared, [D-Mel6]LH-RH (SB-05) and [Ac-D-Nal(2)1,D-Phe(pCl)2,D-Pal(3)3,Arg5,D-Mel6,D-Ala10++ +]LH-RH [SB-86, where Nal(2) is 3-(2-naphthyl)alanine and Pal(3) is 3-(3-pyridyl)alanine] possessed the expected high agonistic and antagonistic activities, respectively, and also showed high affinities for the membrane receptors of rat pituitary cells, human breast cancer cells, human prostate cancer cells, and rat Dunning R-3327 prostate tumor cells. These two analogues exerted cytotoxic effects on human and rat mammary cancer cells in vitro. Thus these two D-Mel6 analogues seem to be particularly suitable for the study of how alkylating analogues of LH-RH could interfere with intracellular events in certain cancer cells.     

Improved stability,insulin-releasing activity and antidiabetic potential of two novel N-terminal analogues of gastric inhibitory polypeptide: N-acetyl-GIP and pGlu-GIP

AIMS/HYPOTHESIS: This study examined the plasma stability, biological activity and antidiabetic potential of two novel N-terminally modified analogues of gastric inhibitory polypeptide (GIP). METHODS: Degradation studies were carried out on GIP, N-acetyl-GIP (Ac-GIP) and N-pyroglutamyl-GIP (pGlu-GIP) in vitro following incubation with either dipeptidylpeptidase IV or human plasma. Cyclic adenosine 3'5' monophosphate (cAMP) production was assessed in Chinese hamster lung fibroblast cells transfected with the human GIP receptor. Insulin-releasing ability was assessed in vitro in BRIN-BD11 cells and in obese diabetic ( ob/ ob) mice. RESULTS: GIP was rapidly degraded by dipeptidylpeptidase IV and plasma (t(1/2) 2.3 and 6.2 h, respectively) whereas Ac-GIP and pGlu-GIP remained intact even after 24 h. Both Ac-GIP and pGlu-GIP were extremely potent ( p<0.001) at stimulating cAMP production (EC(50) values 1.9 and 2.7 nmol/l, respectively), almost a tenfold increase compared to native GIP (18.2 nmol/l). Both Ac-GIP and pGlu-GIP (10(-13)-10(-8) mmol/l) were more potent at stimulating insulin release compared to the native GIP ( p<0.001), with 1.3-fold and 1.2-fold increases observed at 10(-8) mol/l, respectively. Administration of GIP analogues (25 nmol/kg body weight, i.p.) together with glucose (18 mmol/kg) in ( ob/ ob) mice lowered ( p<0.001) individual glucose values at 60 min together with the areas under the curve for glucose compared to native GIP. This antihyperglycaemic effect was coupled to a raised ( p<0.001) and more prolonged insulin response after administration of Ac-GIP and pGlu-GIP (AUC, 644+/-54 and 576+/-51 ng.ml(-1) x min, respectively) compared with native GIP (AUC, 257+/-29 ng.ml(-1) x min). CONCLUSION/INTERPRETATION: Ac-GIP and pGlu-GIP, show resistance to plasma dipeptidylpeptidase IV degradation, resulting in enhanced biological activity and improved antidiabetic potential in vivo, raising the possibility of their use in therapy of Type II (non-insulin-dependent) diabetes mellitus.     

Possible involvement of protein kinase C in gonadotropin and growth hormone release from dispersed goldfish pituitary cells

Static incubation with tumor-promoting 4 beta-phorbol esters, activators of the Ca2(+)- and phospholipid-dependent protein kinase C enzyme (PKC), caused dose-dependent increases in gonadotropin (GTH) and growth hormone (GH) secretion in primary cultures of dispersed goldfish pituitary cells. The estimated half-maximal effective doses (ED50) for stimulating GTH and GH release were 0.35 +/- 0.17 and 0.32 +/- 0.13 nM 12-O-tetradecanoyl phorbol 13 acetate (TPA), 3.71 +/- 1.30 and 1.37 +/- 0.76 nM 4 beta-phorbol 12,13-dibutyrate, 6.90 +/- 4.84 and 1.89 +/- 0.25 nM 4 beta-phorbol 12,13-dibenzoate, and 455 +/- 258 and 311 +/- 136 nM 4 beta-phorbol 12,13-diacetate, respectively. In contrast, treatments with up to 10 microM of the inactive 4 alpha-phorbol 12,13-didecanoate ester did not alter GTH and GH release. Additions of the synthetic diacylglycerol, dioctanoyl glycerol, also enhanced GTH and GH secretion in a dose-dependent manner and with ED50s of 1.73 +/- 0.83 and 1.73 +/- 1.19 microM, respectively. The GTH and GH responses to stimulation by TPA were attenuated by incubation with Ca2(+)-depleted medium containing EGTA or by treatment with the Ca2+ channel blocker verapamil. Coincubation with the PKC inhibitor H7 reduced the GTH and GH responses to TPA. As in previous studies, additions of salmon gonadotropin-releasing hormone (sGnRH) or chicken GnRH-II (cGnRH-II) induced GTH and GH release; these hormone responses to sGnRH and cGnRH-II were also decreased by the addition of H7. These results indicate that activation of PKC may stimulate GTH and GH release in goldfish and suggest that sGnRH and cGnRH-II actions on goldfish pituitary GTH and GH secretion are also mediated, at least partially, by PKC.     

Production of interleukin-6 by anterior pituitary cells in vitro

We recently reported that the cytokine interleukin-6 (IL-6) is a potent stimulator of anterior pituitary hormone release in vitro. Since IL-6 is not normally detectable in the blood, we hypothesized that IL-6 may be produced by the anterior pituitary in situ and thereby affect hormone secretion through paracrine or autocrine mechanisms. The present study demonstrates that cultured anterior pituitary cells spontaneously secrete large quantities of IL-6 in vitro. IL-6 was detectable in the incubation medium within 2 h, and by 8 h of culture had attained concentrations of 2000-4000 U/ml.4 x 10(5) cells. IL-6 production was stimulated by phorbol myristate acetate (10-100 nM) approximately 2-fold and by lipopolysaccharide (0.001-10.0 micrograms/ml) 4-fold during 4-h incubations. In contrast, the cytokine recombinant human IL-1 alpha had no effect on IL-6 release by cultured pituitary cells. Freshly dissected hemipituitary tissue also secreted more than 3000 U/ml IL-6 during a 4-h incubation. This secretion was enhanced 3-fold by 10 micrograms/ml lipopolysaccharide. Our results suggest that the anterior pituitary may produce IL-6 in situ, where it may function as an intrapituitary releasing factor.     

Relative potencies of gonadotropin-releasing hormone agonists and antagonists on ovarian and pituitary functions

The abilities of various agonistic and antagonistic analogs of GnRH to modulate ovarian estrogen production were compared with their relative potencies to regulate pituitary LH release. Granulosa cells from immature hypophysectomized rats were cultured for 2 days in the presence of FSH and aromatase substrate (delta 4-androstenedione), with or without various concentrations of GnRH agonists or 10(-8) M GnRH plus various concentrations of selected GnRH antagonists. FSH treatment increased estrogen production, whereas concomitant treatment with various GnRH agonists resulted in dose-dependent decreases in estrogen production. des-Gly10-[D-Ser(TBu)6] Pro9-NHEt-GnRH proved most potent at both the ovarian and pituitary levels, being 170- and 190-fold greater than those values for the GnRH decapeptide, respectively. In general, the ovarian and pituitary potencies of all 10 agonists studied were comparable, with a correlation coefficient of 0.98. When added together with 10(-8) M GnRH, the GnRH antagonists caused dose-dependent blockage of the inhibitory effect of GnRH. Among 7 antagonists tested, [Ac-D-Phe1,D-p-cl-Phe2,D-TYrp3,6]GnRH was shown to be most potent at the pituitary level (half maximal inhibitory dose ratio: IDR50 antagonist/GnRH = 0.17) and blocked the GnRH inhibition at the ovarian level with an IDR50 value of 4.4. The potencies of the antagonists to block the GnRH effect at the pituitary and ovarian levels were comparable, yielding a correlation coefficient of 0.97. Although one cannot rule out subtle differences in the specificities of the respective GnRH target tissues due to minor disparities between pituitary and ovarian potencies of some analogs, the present results demonstrate an overall agreement of responsiveness between ovarian granulosa cells and pituitary gonadotrophs to GnRH agonists and antagonists.