Superfused Rat Pituitary Cells Research Articles

An evaluation of intravenous, subcutaneous, and in vitro activity of new agmatine analogs of growth hormone-releasing hormone hGH-RH(1–29)NH 2

The effects of new Agmatine (Agm) analogs of human growth hormone-releasing hormone (GH-RH) were compared to GH-RH(1–29)NH 2 and to (D-Ala 2)GH-RH(1–29)NH 2 after intravenous (IV) and subcutaneous (SC) administration to pentobarbital-anesthetised male rats and in vitro using superfused rat pituitary cell system. After IV administration, the analogs: (D-MeAla 2, Nle 27)GH-RH(1–28)Agm(JG-75), (desamino-Tyr 1, D-Ala 2, Nle 27)GH-RH(1–28)Agm(JG-77), (D-Ala 2, Nle 27)GH-RH(1–28)Agm(JG-73) and (D-Ala 2)GH-RH(1–29)NH 2 showed a potency 2.6–3.9 times greater than GH-RH(1–29)NH 2 at 5 min and 1.6–2.7 times higher at 15 min. After SC administration these analogs were 30–74 times more potent than GH-RH(1–29)NH 2. The ratio between the IV and SC GH-releasing activity of the analogs ranged from 2 to 5, while GH-RH(1–29)NH 2 was about 50 times more active IV than SC. This indicates that 20–50% of the analogs can be absorbed from SC tissues, but only 2% of GH-RH(1–29)NH 2. The in vitro activity of the agmatine analogs on GH release closely paralleled their IV potency and was 2.8–3.9 times greater than that of GH-RH(1–29)NH 2. No significant difference in potency was found between (D-Ala 2)GH-RH(1–29)NH 2 and JG-75 after IV administration and in vitro, although JG-75 contained only 28 amino acids. We conclude that the reason for the large discrepancies between the previously reported activities of (D-Ala 2)GH-RH(1–29)NH 2 was simply due to the different ways of administration of this analog, SC vs IV, and not to species specificity. The replacement of Arg 29 by Agmatine in (D-Ala 2, Nle 27)GH-RH(1–29)NH 2 causes a 3 fold increase in SC potency, but the replacement of D-Ala 2 with D-MeAla 2 reduces the SC, but not the IV and in vitro activity in half.

Synthesis, purification and biological evaluation of porcine corticotropin-releasing factor

The 41-residue sequences of recently identified porcine corticotropin-releasing factors [I1e 40]pCRF and [Asn 40]pCRF were assembled on a benzhydrylamine resin support. Deprotection and cleavage from the resin were accomplished by HF treatment. The crude peptides were purified by HPLC. The homogeneity of the final materials, obtained in 0.2% and 0.4% overall yield for [I1e 40]pCRF and [Asn 40]pCRF respectively, was assessed after the isolation by HPLC and amino acid analysis. Both sequences of the synthetic 41-residue pCRF stimulated the release of corticotropin (ACTH) from superfused rat pituitary cells on a column, the responses being related to a log-dose of CRF in the range of 1–20 ng/ml. [I1e 40]pCRF and [Asn 40]pCRF also augmented the in vivo release of ACTH in rats pretreated with chlorpromazine, morphine and Nembutal. [I1e 40]pCRF appeared to be equipotent to ovine CRF and about twice as active as [Asn 40]pCRF. The data indicate that synthetic porcine [I1e 40]pCRF and [Asn 40]pCRF have high biological activity.

Arachidonic acid and its lipoxygenase metabolites stimulate prolactin release in superfused pituitary cells.

The direct effect of leukotrienes and other lipoxygenase products on prolactin release has been assessed. Arachidonic acid and its lipoxygenase metabolites 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) and 15-hydroxy-5,8,10,14-eicosatetranoic acid (15-HETE) stimulated the release of prolactin in superfused rat pituitary cells in a dose-dependent manner. Leukotrienes (LT) A4, B4, C4 and E4 provoked a very marked biphasic and dose-dependent secretion of prolactin from superfused cells. Maximal effects were achieved with leukotrienes at a concentration of 3 X 10(-11) to 3 X 10(-10) M but LTD4 did not affect peptide release under these conditions. The metabolites were more potent than arachidonic acid in affecting hormone secretion. Pulses of 4 minutes duration of these fatty acids may even elicit a more pronounced response than thyrotrophin-releasing hormone (TRH). Nordihydroguaiaretic acid (NDGA 10(-6) M), a lipoxygenase inhibitor, prevented the effect of arachidonic acid on peptide secretion. Repeated TRH (10(-7) M) administration to pituitary cells led to a reduction in cell response, which may also be observed in cells pre-treated with pulsatile 5-HETE or 15-HETE. These data support previous findings that arachidonic acid and its lipoxygenase metabolites may play a role in the secretory mechanism of prolactin release in pituitary cells.

Stimulation of gonadotropin release by arachidonic acid and its lipoxygenase metabolites in superfused pituitary cells

Luteinizing hormone and follicle stimulating hormone secretion was stimulated by 4 min pulses of arachidonic acid (3×10 −5 to 10 −4M) in superfused rat pituitary cells. The effect of its lipoxygenase metabolites, 5-hydroxy-6,8,11,14-eicosatetranoic acid (5-HETE) and 15-hydroxy-5,8,10,14-eicosatetranoic acid (15-HETE) was more potent on hormone release when added in the same dose. Using 3×10 −5M 5-HETE, its releasing activity on gonadotropins was comparable to that of GnRH (10 −9M). 15-HETE (3×10 −5M) was even more potent on LH and FSH secretion than 5-HETE. The secretory profile induced by 5-HETE and 15-HETE was also similar to that shown for GnRH, resulting in a rapid increase and a more prolonged decline of the hormone release. The addition of these fatty acids to superfused pituitary cells did not alter the response of the cells to their physiological ligand. These findings give further support to the proposal that metabolites of arachidonic acid may be involved in receptor-mediated mechanisms of gonadotropin release in pituitary cells.

Comparison of in vitro and in vivo effects of different species specific GnRH and their analogs

Mammalian, salmon and chicken gonadotropin-releasing hormones (mGnRH, sGnRH, cGnRH) and their analogs were synthesized and tested for their ability to stimulate in vitro LH and FSH release from cultured and superfused rat pituitary cells and also their in vivo effect were investigated on the artificial propagation of fishes. The LH and FSH releasing activity of sGnRH, cGnRH and their analogs were lower than the appropriate mammalian ones from cultured rat pituitary cells, but two of the cGnRH analogs showed increased LH and FSH secretory activity from superfused rat pituitary cells compared to the mGnRH. At the same time these two analogs are very potent to stimulate reproductive function of fishes and using these peptides we were able to fulfill the artificial propagation of fishes which could not be artificially propagated before.

Effect of atrial natriuretic peptide on gonadotropin release in superfused rat pituitary cells.

Cardiac atrial muscle cells produce a polypeptide hormone that plays a role in the control of water and electrolyte balance and blood pressure. The circulating form of this hormone is the atrial natriuretic peptide (ANP), which contains 28 amino acids. Various immunohistochemical studies have shown that ANP is present in many areas of the central nervous system, including the median eminence. In our studies, we investigated the effect of ANP in a superfused rat pituitary cell system. When ANP was administered at increasing concentrations (0.01 microM to 1 microM), it caused a significant dose-related stimulation of the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). The lowest effective dose of ANP in our system was 0.03 microM. When ANP and LH-releasing hormone were administered together, the response was prolonged and had the characteristics of ANP-stimulated LH and FSH release. In contrast with some previous reports, ANP in high concentration (1 microM) consistently induced a small but significant stimulation of the release of corticotropin. ANP did not influence the basal release of prolactin, growth hormone, and thyrotropin.

Purification and characterization of peptides with corticotropin-releasing factor activity from porcine hypothalami.

Ten polypeptides that stimulated the release of corticotropin from superfused rat pituitary cells and that are structurally related to porcine corticotropin-releasing factor were isolated from porcine hypothalami. The purification was carried out by gel filtration followed by reversed-phase HPLC using trifluoroacetic acid or heptafluorobutyric acid as the ion-pairing agent in water/acetonitrile solvent systems. The purified peptides were homogeneous by chromatography and by sequence analysis. One major polypeptide was characterized. Its structure is -H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Gl u-Val -Leu-Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys -Leu-Met-Glu-Asn-Phe-NH2 [Patthy, M., Horvath, J., Mason-Garcia, M., Szoke, B., Schlesinger, D. H. & Schally, A. V. (1985) Proc. Natl. Acad. Sci. USA 82, 8762-8766]. This 41-amino acid sequence is thought to represent porcine corticotropin-releasing factor. Based on automated gas-phase sequencing of the intact and CNBr-cleaved peptides, amino acid analysis, and carboxypeptidase Y digestion, the other nine polypeptides were found to be structurally similar to this 41-amino acid sequence. Modifications of this structure include deamidation of glutamine at position 26 or 29, oxidation of methionine at positions 21 and/or 38, a blocked N terminus, and deletion of phenylalanine amide at the C terminus. Eight of these nine modified peptides retained significant corticotropin-releasing factor activity as shown by the stimulation of corticotropin release from superfused rat and pig pituitary cells. Some of these peptides may be present in pig hypothalami, while the others could have been produced during the isolation.

Isolation and amino acid sequence of corticotropin-releasing factor from pig hypothalami.

A polypeptide was isolated from acid extracts of porcine hypothalami on the basis of its high ability to stimulate the release of corticotropin from superfused rat pituitary cells. After an initial separation by gel filtration on Sephadex G-25, further purification was carried out by reversed-phase HPLC. The isolated material was homogeneous chromatographically and by N-terminal sequencing. Based on automated gas-phase sequencing of the intact and CNBr-cleaved peptide and on carboxypeptidase Y digestion, the primary structure of this 41-residue polypeptide was determined to be Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val -Leu-Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys -Leu-Met-Glu-Asn-Phe-NH2. Porcine corticotropin-releasing factor (CRF) shares a common amino acid sequence (residues 1-39) with rat and human CRF and differs from these only in positions 40 and 41. However, isoleucine was also present at position 40 in porcine CRF, but in a smaller percentage than asparagine. The sequence of porcine CRF shows 83% homology with ovine CRF. Porcine CRF markedly stimulated the release of corticotropin from superfused rat and pig pituitary cells. The biological activity and close structural relationship to CRFs of other species indicate that the peptide isolated represents porcine CRF.