Superfused Rat Pituitary Cells Research Articles

Direct anticancer activity of gonadotropin-releasing hormone-III.

In previous studies GnRH-III, a variant of the hypothalamic neurohormone GnRH, was isolated from the brain of the sea lamprey and structurally characterized. GnRH-III is a hypothalamic neurohormone in both female and male sea lampreys. In the present work biological activities of GnRH-III in mammalian systems were examined. In superfused rat pituitary cells, GnRH-III at 1 nM to 100 nM neither induced LH-secretion nor inhibited the LH-secretion elicited by native GnRH and elicited LH release only at 1 microM. At high dose (500 microg/day) in vivo, GnRH-III behaved as a GnRH agonist, though, it was 1000-fold less active than ovurelin. The in vitro and in vivo results were in good agreement in showing that GnRH-III is only a weak agonist of the endocrine activity of GnRH. GnRH-III specifically bound to receptors on cancer cells and recognized not only the high-, but also the low-affinity binding sites. GnRH-III significantly suppressed growth of human cancer cells which have GnRH receptors. The inhibitory effect of GnRH-III on growth of cancer cells was specific and direct since the peptide did not have endocrine activity in the concentration range found to be effective in anticancer assays. GnRH-III inhibited equally the growth of ER-positive and -negative breast and TeR-positive and negative prostate cells.

Inhibition of GH Release in Rats by New Potent Antagonists of Growth Hormone-Releasing Hormone (GH-RH)

KovÁCs, M., A. V. Schally, M. ZarÁNdi and K. Groot. Inhibition of GH release in rats by new potent antagonists of growth hormone-releasing hormone (GH-RH). Peptides 18(3) 431–438, 1997.—Biological activity of a new series of potent GH-RH antagonists containing formyl or phenylacetyl group at the N-terminus of the sequence [d-Arg2,Phe(4-Cl)6,Nle27]hGH-RH(1-29)NH2, as well as various substitutions in positions 8, 15, or 28, and in some cases Agm in position 29, was evaluated in vivo. All five antagonists, administered at a 27-fold molar excess to rats, suppressed the GH-releasing effect of exogenous GH-RH(1-29)-NH2 by 64–75%. The inhibitory effects lasted for more than 15 min. The most potent analogue, PhAc-[d-Arg2,Phe(4-Cl)6,Abu15,Nle27]hGH-RH(1-28)Agm (MZ-5-156), showed an in vivo potency 7–16 times higher than the early antagonist [Ac-Tyr1,d-Arg2]hGH-RH(1-29)-NH2, which was used as standard. MZ-5-156 was capable of decreasing serum GH levels after intravenous, intraperitoneal, or intramuscular administration. In vitro, in the superfused rat pituitary cell system, MZ-5-156 induced a prolonged inhibition of GH release after continuous long-term administration and showed a potency more than 100 times greater than the standard antagonist. These results show that N-terminal acylation with phenylacetic acid of the sequence [d-Arg2,Phe(4-Cl)6,Nle27]hGH-RH(1-29)-NH2, containing modifications in positions 8, 15, 28, or 29, results in antagonists with high and protracted potency both in vivo and in vitro. In view of high antagonistic activity and prolonged duration of action, some of these antagonists of GH-RH may find clinical application for the treatment of IGF-dependent cancers.

Synthesis and In Vitro Evaluation of New Potent Antagonists of Growth Hormone-Releasing Hormone (GH-RH)

Zarandi, M., M. Kovacs, J. E. Horvath, K. Toth, G. Halmos, K. Groot, A. Nagy, Z. Kele, and A. V. Schally, Synthesis and in vitro evaluation of new potent antagonists of growth hormone-releasing hormone (GH-RH). Peptides 18(3) 423–430, 1997.—In the search for more potent antagonists of hGH-RH, 20 new analogs were synthesized, purified and tested in vitro. All the analogs were based on the N-terminal sequence of 28 or 29 amino acid residues of hGH-RH, but contained D-Arg2 and Nle27 modifications. Most analogs had Phe (pCl)6 and Agm29 substituents. The effect of other substitutions such as Abu8 and/or Abu15 and Ala 15 and various hydrophobic and hydrophilic d or l amino acids at position 8 were also investigated. All the peptides were acylated at the N-terminus in an attempt to increase the antagonistic activity. In the superfused rat pituitary cell system, most analogs inhibited more powerfully the GH release induced by GH-RH than the standard antagonist [Ac-Tyr1, D-Arg2]hGH-RH (1-29)-NH2. Some antagonists were long acting. Among the peptides synthesized, antagonist PhAc-[D-Arg2, Phe (pCl)6, Abu15, Nle27] hGH-RH (1-28)Agm (MZ-5-156) appeared to be the most potent and inhibited GH release in vitro 63-200 times more powerfully than the standard antagonist. MZ-5-156 and other antagonists showed high binding affinities to membrane receptors for GH-RH. Some of these hGH-RH antagonists could be further developed for possible onocological applications.

Effects of acute and chronic administration of a new potent antagonist of growth hormone-releasing hormone in rats: mechanisms of action.

Antagonistic analogs of human GH-releasing hormone (hGHRH) are potential candidates for the treatment of disorders characterized by excessive GH secretion and especially for therapy of GH- and insulin-like growth factor (IGF)-dependent tumors. These analogs should be also useful for the studies on the mechanism of action of GHRH. In the present investigation, we evaluated the effects of chronic i.m. administration of a new potent GHRH antagonist (Ibu0,D-Arg2,Phe(4-Cl)6,Abu15,Nle27)hGHRH(1-28)+ ++Agm (MZ-4-71) on the growth rate, serum GH, and IGF-I concentration, GH responsiveness to exogenous GHRH, as well as the pituitary GH content and GHRH receptor concentration in young female rats. We also studied the consequences of acute, high-dose i.v. application of this antagonist on the basal GH and IGF-I levels in adult male rats. In addition, the ability of GHRH antagonist MZ-4-71 to prevent GH release induced by GHRH pulses was determined in vitro in the superfused rat pituitary cell system. Chronic treatment in vivo using twice daily i.m. injections of 20 microg MZ-4-71 for 2 weeks reduced the rate of increase in body weight by 21% and in body length of young rats by 36%, as compared with controls. GH responses to bolus injections of GHRH declined by 22%, and serum IGF-I concentrations by 15% at the end of the treatment. The total pituitary GH content, but not relative GH concentration, also decreased by 15% and GHRH receptor concentration by 48%, following chronic treatment with this antagonist. Bolus injections of high doses of MZ-4-71 (400 microg i.v.) induced a marked and protracted (6 h) inhibition of the basal serum GH concentration and a parallel inhibition of the serum IGF-I levels. The nadir of both the serum GH (62% decrease) and the IGF-I level (30% decrease) was found at 3 h after the injection. In vitro studies showed that MZ-4-71 was able to dose-dependently inhibit the GH-releasing effect of GHRH pulses. Present results demonstrate that GHRH antagonist MZ-4-71 is effective in vivo and that it can inhibit growth and secretion of GH and IGF-I in rats. Our findings also provide new information on the role of GHRH in regulating synthesis of GH and GHRH receptors. It is likely that antagonistic analogs of GHRH could find clinical application for reducing the growth of tumors dependent on GH or IGF-I.

Ca2+ entry in gonadotrophs and αT3–1 cells: does store-dependent Ca2+ influx mediate gonadotrophin-releasing hormone action?

In pituitary gonadotrophs GnRH causes biphasic (spike and plateau) increases in cytosolic Ca2+ ([Ca2+]i) and gonadotrophin release. The spike phases reflect mobilization of stored Ca2+ and the plateau responses are attributed, in part, to Ca2+ influx via voltage-sensitive Ca2+ channels. In recent years, store-dependent Ca2+ influx (SDCI), in which depletion of the intracellular inositol 1,4,5-trisphosphate-mobilizable pool stimulates Ca2+ influx, has emerged as a major form of Ca2+ entry activated by phosphoinositidase C-coupled receptors in non-excitable cells. More recent evidence also indicates a role for SDCI in excitable cells. We have used dynamic video imaging of [Ca2+]i in alpha T3-1 cells (a gonadotroph-derived cell line) and manipulation of the filling state of the GnRH-mobilizable Ca2+ pool to test the possible role of SDCI in GnRH action. In Ca(2+)-containing medium, GnRH caused a biphasic increase in [Ca2+]i whereas in Ca(2+)-free medium only a transient increase occurred. The response to a second stimulation with GnRH in Ca(2+)-free medium was reduced by > 95% (demonstrating that Ca2+ pool depletion had occurred) and was recovered after brief exposure to Ca(2+)-containing medium (which enables refilling of the pool). Ionomycin (a Ca2+ ionophore) and thapsigargin (which inhibits the Ca(2+)-sequestering ATPase of the endoplasmic reticulum) also transiently increased [Ca2+]i in Ca(2+)-free medium and depleted the GnRH-mobilizable pool as indicated by greatly reduced subsequent responses to GnRH. Pool depletion also occurs on stimulation with GnRH in Ca(2+)-containing medium because addition of ionomycin and Ca(2+)-free medium during the plateau phase of the GnRH response caused only a reduction in [Ca2+]i rather than the transient increase seen without GnRH. To deplete intracellular Ca2+ pools, cells were pretreated in Ca(2+)-free medium with thapsigargin or GnRH and then, after extensive washing, returned to Ca(2+)-containing medium. Pretreatment with thapsigargin augmented the increase in [Ca2+]i seen on return to Ca(2+)-containing medium (to two- to threefold higher than that seen in control cells) indicating the activation of SDCI, whereas pool depletion by GnRH pretreatment had no such effect. To ensure maintained pool depletion after Ca2+ re-addition, similar studies were performed in which the thapsigargin and GnRH treatments were not washed off, but were retained through the period of return to Ca(2+)-containing medium. Return of GnRH-treated cells to Ca(2+)-containing medium caused an increase in [Ca2+]i which was inhibited by nicardipine, whereas the increase seen on return of thapsigargin-treated cells to Ca(2+)-containing medium was not reduced by nicardipine. The quench of fura-2 fluorescence by MnCl2 (used as a reporter of Ca2+ influx) was increased by GnRH and thapsigargin, indicating that both stimulate Ca2+ influx via Mn2+ permeant channels. The GnRH effect was abolished by nicardipine whereas that of thapsigargin was not. Finally, depletion of intracellular Ca2+ pools by pretreatment of superfused rat pituitary cells with GnRH or thapsigargin in Ca(2+)-free medium did not enhance LH release on return to Ca(2+)-containing medium. The results indicate that (a) thapsigargin stimulates SDCI in alpha T3-1 cells via nicardipine-insensitive Ca2+ channels, (b) in spite of the fact that GnRH depletes the hormone-mobilizable Ca2+ pool, it fails to stimulate SDCI, (c) GnRH stimulates Ca2+ entry predominantly via nicardipine-sensitive channels, a route not activated by SDCI and (d) in rat gonadotrophs, GnRH-stimulated LH release is not mediated by SDCI.

Effect of long-acting antagonists of growth hormone (GH)-releasing hormone on GH and cyclic adenosine 3',5'-monophosphate release in superfused rat pituitary cells

Effect of long-acting antagonists of growth hormone (GH)-releasing hormone on GH and cyclic adenosine 3',5'-monophosphate release in superfused rat pituitary cells

Effect of long-acting antagonists of growth hormone (GH)-releasing hormone on GH and cyclic adenosine 3',5'-monophosphate release in superfused rat pituitary cells.

In a recent study, investigating the time course of both GH and cAMP secretion induced by GH-releasing hormone (GHRH) in the superfusion system, we found that the amount of cAMP liberated from the cells was not proportional to GH release and that cAMP discharged after a GHRH pulse alone cannot maintain the release of GH. In the present study, two potent antagonists of GHRH, MZ-4-71 ([Ibu0,D-Arg2,Phe(4-Cl)6,Abu15,Nle27]human GHRH-(1-28)Agm) and MZ-4-243 ([Nac0,D-Arg2,Phe(4-Cl)6,Abu15,Nle27]human GHRH-(1-28)Agm) were evaluated for their long term effect in the superfusion system and for their ability to influence the release of GH and cAMP. Our present findings showed that after a 9-min preincubation, antagonist MZ-4-71 and MZ-4-243 at 3 and 1 nM, respectively, caused an inhibition of GH release, stimulated by 1 nM GHRH, similar to that caused by the 100-nM dose of the standard antagonist ([Ac-Tyr1,D-Arg2]human GHRH-(1-29)NH2). The standard antagonist at 100 nM did not influence the GHRH-induced GH response 30 min later, and the inhibition caused by MZ-4-71 at 30 nM decreased gradually to 30% 120 min after the treatment, but the 30-nM dose of MZ-4-243 reduced the GH response by more than 90% even 270 min after its administration. During a 2-h incubation with 1 nM GHRH in combination with a 30-min infusion of the standard antagonist, MZ-4-71, MZ-4-243, or somatostatin, from the 30th to the 60th min, the decrease in GH discharge preceded the inhibition of cAMP release. After infusion of the antagonists or somatostatin was stopped, GH release resumed sooner than that of cAMP. Simultaneous determinations of cAMP and GH in the samples showed that changes in GH levels were never preceded by a rise or decrease in cAMP release, in contrast to existing information. The participation of more than one signal transduction mechanism in the mediation of the effect of GHRH is very likely, and the balance of these mechanisms may vary with the dose and duration of stimulation.

Synthesis and biological evaluation of superactive agonists of growth hormone-releasing hormone.

Analogs of the 29 amino acid sequence of human growth hormone-releasing hormone (hGH-RH) with agmatine (Agm) in position 29, desaminotyrosine (Dat) in position 1, norleucine (Nle) in position 27, and L-alpha-aminobutyric acid (Abu) in position 15 have been synthesized, and their biological activity was evaluated. Some peptides contained one or two residues of ornithine (Orn) instead of Lys in positions 12 and 21 and additional replacements in positions 8 and 28. All analogs were found to be more potent than hGH-RH-(1-29)-NH2 in the superfused rat pituitary cell system. In tests in vivo in rats after subcutaneous administration, the analogs JI-22, [Dat1, Orn12,21, Abu15, Nle27, Agm29]hGH-RH-(1-29); JI-34, [Dat1, Orn12,21,Abu15,Nle27, Asp28, Agm29]hGH-RH-(1-29); JI-36, [Dat1, Thr8, Orn12,21, Abu15,Nle27,Asp28,Agm29]hGH-RH-(1-29); and JI-38, [Dat1,Gln8, Orn12,21,Abu15,Nle27,Asp28,Agm29]hGH-RH-(1 -29) displayed a potency 44.6,80.9,95.8, and 71.4 times greater, respectively, than that of hGH-RH-(1-29)-NH2 at 15 min and 217.1, 89.7, 87.9, and 116.8 times greater at 30 min. After intravenous administration, JI-22, JI-36, and JI-38 were 3.2-3.8 times more potent than hGH-RH-(1-29)-NH2 at 5 min and 6.1-8.5 times more active at 15 min. All analogs were found to have higher binding affinities for GH-RH receptors on rat pituitary cells than hGH-RH-(1-29)-NH2. Because of high activity and greater stability, these analogs could be considered for therapy of patients with growth hormone deficiency.

Growth hormone-releasing hormone stimulates cAMP release in superfused rat pituitary cells.

The release of growth hormone (GH) and cAMP was studied in superfused rat pituitary cells by infusing growth hormone-releasing hormone (GHRH) at different doses or a combination of GHRH and somatostatin 14 (SS-14). Three-minute pulses of GHRH caused a dose-dependent GH and cAMP release (effective concentration of 50% of the maximal biological effect is 0.21 nM and 52.5 nM, respectively). The lowest effective doses of GHRH in the superfusion system were 0.03 nM for GH release and 0.3 nM for cAMP discharge when 3-min pulses were applied. The amount of cAMP liberated from the cells was not proportional to GH release: cAMP responses to low doses of GHRH were disproportionally small, and the gradual increase in the release of cAMP after high doses of GHRH was not followed by a parallel rise in GH release. The desensitization induced by repeated pulses or prolonged infusion of GHRH resulted in a greater reduction in GH release than in cAMP liberation. A simultaneous infusion of SS-14 completely blocked GH release stimulated by GHRH but did not inhibit the immediate release of cAMP caused by GHRH. An abrupt decrease in GHRH-stimulated GH release induced by SS-14 was followed by only a minimal reduction in cAMP liberation 9 min later. Our findings indicate that a discharge of cAMP is stimulated after a GHRH pulse, but this effect alone cannot maintain the release of GH. Other steps of the signal transduction mechanisms that are independent of the cAMP route may participate in the process of GH release. The nature of the mechanisms involved in the mediation of GH release may vary with the doses of GHRH used.

Synthesis and biological activities of highly potent antagonists of growth hormone-releasing hormone.

In the search for antagonists of human growth hormone-releasing hormone (hGHRH) with high activity, 22 analogs were synthesized by solid-phase methods, purified, and tested biologically. Within the N-terminal sequence of 28 or 29 amino acids of hGHRH, all the analogs contained D-Arg2, Phe(4-Cl)6 (para-chlorophenylalanine), Abu15 (alpha-aminobutyric acid), and Nle27 and most of them had Agm29 (agmatine) substituents. All the peptides, except one, were acylated at the N terminus with different hydrophobic acids--e.g., isobutyric acid (Ibu) or 1-naphthylacetic acid (Nac) in order to study the effect of N-terminal acylation on the antagonistic activity. In the superfused rat pituitary cell system, all the analogs inhibited more powerfully the GHRH-induced growth hormone (GH) release than the standard GHRH antagonist [Ac-Tyr1,D-Arg2]hGHRH-(1-29)NH2. Antagonists [Ibu0,D-Arg2,Phe(4-Cl)6,Abu15,Nle27]hGHRH-(1-28) Agm (MZ-4-71), [Nac0,D-Arg2,Phe(4-Cl)6,Abu15,Nle27]hGHRH-(1-28) Agm (MZ-4-243), [Nac0,D-Arg2,Phe(4-Cl)6,Abu15,Nle27]hGHRH-(1-29) NH2 (MZ-4-169), [Nac0-His1,D-Arg2,Phe(4-Cl)6,Abu15,Nle27]-hGH RH-(1-29)NH2 (MZ-4-181), and [Nac0,D-Arg2,Phe(4-Cl)6,Abu15,Nle27,Asp28]hGH RH-(1-28)Agm (MZ-4-209) inhibited GH release at 3 x 10(-9) M. Among these peptides, MZ-4-243, MZ-4-169, and MZ-4-181 were also long acting in vitro. Antagonist MZ-4-243 inhibited GH release 100 times more powerfully than the standard antagonist and was the most potent in vitro among GHRH antagonists synthesized. Analogs with high inhibitory effects in vitro were also found to have high affinities to rat pituitary GHRH receptors. In experiments in vivo, antagonists [Ibu0,D-Arg2,Phe(4-Cl)6,Abu15,Nle27]-hGHRH-(1-28 )Agm (MZ-4-71), [Nac0,D-Arg2,Phe(4-Cl)6,Abu15,Nle27]hGHRH-(1-29) NH2 (MZ-4-169), and [Nac0-His1,D-Arg2,Phe(4-Cl)6,Abu15,Nle27]hGHR H-(1-29)NH2 (MZ-4-181) induced a significantly greater inhibition of GH release than the standard antagonist. In view of their high antagonistic activity and prolonged duration of action, some of these antagonists of GHRH may find clinical applications, including treatment of certain endocrine disorders and insulin-like growth factor I-dependent tumors.

LH-RH analogue carrying a cytotoxic radical is internalized by rat pituitary cells in vitro

The binding and internalization of a cytotoxic analogue of luteinizing hormone-releasing hormone (LH-RH), T-98 (agonist [ d-Lys 6]LH-RH linked to glutaryl-2-(hydroxymethyl)anthraquinone), by rat anterior pituitary cells was investigated. Analogue T-98 was bound to pituitary membrane binding sites for LH-RH with a high affinity ( K d =1.2 nM ) and was 17 times more potent in releasing luteinizing hormone (LH) from superfused rat pituitary cells than LH-RH. The labeling of this cytotoxic LH-RH analogue was carried out both with radioactive ( 125I) and nonradioactive iodine. Monoiodination of the Tyr 5 residue of T-98 did not significantly affect its binding affinity but greatly decreased its LH-releasing activity to about 3% of the original value. Di-iodination in the same position lowered binding affinity twenty-threefold and further diminished LH-releasing potency. [ 125I]T-98 was found to bind very strongly to polystyrene, which precluded the use of regular tissue culture plasticware in our experiments. In pituitary cells cultured in glass vials, binding and internalization of [ 125I]T-98 were observed, which were time and temperature dependent, and which could be inhibited by excess unlabeled analogue. No enzymatic degradation of labeled T-98 was detected in the culture medium during the incubation. Our results indicate that T-98 is internalized by pituitary gonadotropes through receptor-mediated endocytosis. Because this new class of compounds was designed as anticancer drugs, our findings also suggest that this cytotoxic LH-RH agonist may also be internalized by LH-RH receptors present in breast, prostate, ovarian, and other tumors.

Novel antitumor peptide hormones and their effect on signal transduction

A series of novel gonadotropin releasing hormone (GnRH) and Somatostatin analogs have been developed in our laboratory and were screened for antiproliferative and signal transduction inhibitory effect. Our GnRH analog Folligen, had significant antitumor activity on DMBA induced mammary carcinomas in rats without blocking ovarian functions. The direct effect of Folligen and Buserelin has been compared on the human breast cancer cell line MDA-MB-231. Folligen was found to be more effective in inhibiting cell proliferation and significant differences were found in the signal transduction pathways activated by these analogs. Our novel Somatostatin analogs were screened for tyrosine kinase inhibition and for antiproliferative effect on human colon tumor cells and for growth hormone (GH) release inhibition in vitro and in vivo. The analog TT-2-50 was significantly more active inhibiting GH release in superfused rat pituitary cells and in vivo than native Somatostatin and it strongly inhibited tyrosine kinase and proliferation while it stimulated protein kinase C activity.

Involvement of dopamine D1 receptors in the control of growth hormone secretion in the rat

The effects of dopamine on GH release were investigated both in vivo in freely moving intact rats and in rats with a mediobasal hypothalamic lesion, and in vitro in a perifusion system using dispersed male rat pituitary cells kept in primary culture. In vivo, dopamine (5 mg/kg body weight) induced a rapid and very transient increase in plasma GH levels in lesioned but not in intact rats. This increase was markedly inhibited by a prior injection of the D1 antagonist SCH 23390 (0.5 mg/kg) but not of the D2 antagonist domperidone (0.5 mg/kg). The D1 agonist SKF 38393 induced a dose-dependent stimulation of GH release in lesioned rats, and the effect obtained with a dose of 5 mg/kg was abolished by pretreatment with SCH 23390 (0.5 mg/kg). In vitro, dopamine (0.1 mumol/l) and SKF 38393 (0.1 mumol/l) provoked a rapid and reversible release of GH from superfused rat pituitary cells; this effect was markedly inhibited by simultaneous superfusion of SCH 23390 (1 mumol/l). These findings indicate that dopamine can stimulate basal GH release at the pituitary level and that this stimulation is mediated by D1 but not by D2 receptors. They also support the hypothesis that unidentified hypothalamic neurohormones may modulate this effect.

Action and formation of inositol bisphosphate and inositol trisphosphate in rat anterior pituitary cells

Inositol 4,5-bisphosphate and inositol 1,4,5-trisphosphate, administered exogenously at a concentration of 3 x 10(-5) mol/l increased LH release in superfused rat pituitary cells by 950 +/- 267% and 281 +/- 83%, respectively. This stimulatory effect was reversible and dose-dependent. Other inositol phosphates (inositol 1-monophosphate, inositol 1,4,5,6-tetrakisphosphate, inositol 1,3,4,5,6-pentakisphosphate and inositol 1,2,3,4,5,6-hexakisphosphate), tested in vitro, did not significantly influence LH release. In saponin-permeabilized cells, the rate of basal and stimulated LH release was twice that in non-permeabilized cells. Penetration of inositol bisphosphate and inositol trisphosphate into saponin-treated pituitary cells did not increase the secretory potency of these agents compared with their effect on non-permeabilized cells. The new findings document that inositol trisphosphate formation occurs within 5-45 s after GnRH (10(-7) mol/l) administration and seems to be involved in mediating the rapid, first phase of LH release, whereas inositol bisphosphate formation occurs after 3-15 min and is probably related to later phases of LH secretion. Our results suggest that inositol bisphosphate and inositol trisphosphate are important regulators of the release of luteinizing hormone and can exert their effects not only intracellularly, but also extracellularly.

Action Kinetics of Inhibin in Superfused Pituitary Cells Depend on Gonadotropin-Releasing Hormone Treatment*

We examined the effects of partly purified inhibin from porcine follicular fluid on FSH and LH release in superfused rat pituitary cell cultures exposed to different GnRH stimuli. Pituitary cells from immature male rats were cultured in chemically defined medium. After 4 days of static culture in the absence of inhibin preparation and GnRH, the cell monolayers were superfused for approximately 10 h at a constant speed (0.15 or 0.25 ml/min) with medium with or without inhibin preparation (1 micrograms/ml). During the superfusion, some cultures were stimulated with GnRH (10 nM) continuously or intermittently (1 min/0.5 h or 6 min/1 h). In the basal condition (no GnRH), inhibin suppressed FSH release after 5 h of exposure (P less than 0.01), whereas LH secretion was not affected. In cultures treated with GnRH pulses (of either frequency), the inhibitory effects on the GnRH-stimulated FSH and LH release were statistically significant (P less than 0.01) after 2 h of exposure, became more pronounced in the next several hours, then remained stable until the end of the experiment. In cultures exposed to GnRH continuously, the suppressing effects of inhibin preparation became significant (P less than 0.01) after 3 h of exposure and were maximal at 4 h (52% and 61% of control values for FSH and LH, respectively). Later, the suppressing effect became less pronounce due to the decreasing rate of gonadotropin secretion in control (no inhibin) cultures exposed continuously to GnRH. The magnitude of FSH and LH suppression after 9 h of exposure to the inhibin preparation was statistically different (P less than 0.05) for different GnRH treatments and was more pronounced with GnRH pulses (24-27% and 54-57% of control values for FSH and LH, respectively) than with cultures exposed to GnRH continuously (77% and 89% of control values for FSH and LH, respectively) or in the absence of GnRH (50% and 92% of control values for FSH and LH, respectively). We conclude that both the kinetics and magnitude of action of the inhibin preparation on FSH and LH release can differ significantly depending on the presence or absence of GnRH as well as on the mode of GnRH stimulation. Of particular importance is the observation that suppressive effects of inhibin preparation decline in cultures that have been desensitized to GnRH after prolonged continuous GnRH exposures. These differences stress the role of GnRH-inhibin interactions in the regulation of gonadotropin secretion and emphasize the importance of the mode of GnRH stimulation in studies concerning inhibin action on pituitary cells in vitro.

Estrogen Modulated Gonadotropin Release in Relation to Gonadotropin-Releasing-Hormone (GnRH) and Phorbol Ester (PMA) Actions in Superfused Rat Pituitary Cells*

The involvement of protein kinase C (PKC) in GnRH action is still a matter of controversy. We have conducted a comparative study of LH and FSH release in response to GnRH and to phorbol ester myristate acetate (PMA), an activator of PKC, by rat pituitary cells maintained in culture. The effect of E2 pretreatment coupled or not with PKC depletion was also studied. Different kinetics in the response of LH and FSH to GnRH were observed, suggesting that the intracellular pathways involved in the release process of the two hormones were somewhat different. Moreover, PMA (10 nM) stimulated LH release greatly and FSH release only slightly. Intracellular PKC depletion, obtained by a prolonged treatment (18 h) of the cells with PMA (1 microM), produced different results according to the endocrine status of the pituitary cells. GnRH (10 nM)-induced LH release was significantly decreased in PKC-depleted cells from proestrous females. For PKC-depleted cells from OVX females, it was decreased significantly only when cells had been pretreated by E2. These results suggest that the modulation of LH secretion by E2 involves PKC activation. FSH release was poorly stimulated by PMA; but, under any conditions, PKC depletion did not affect GnRH-induced FSH release.

Oxytocin Is the Major Prolactin Releasing Factor in the Posterior Pituitary*

Although the posterior pituitary is known to contain the PRL releasing activity or factor (PRF), its chemical identification has been a matter of dispute. In the present study, we purified PRF in porcine posterior pituitary extracts to chemically determine the primary structure. PRF activity was assessed during purification by the release of immunoreactive PRL from superfused rat pituitary cells. Two hundred seventy porcine posterior pituitaries were boiled, homogenized, and extracted with 2 M acetic acid. The acid extract was precipitated with 67% acetone, and the supernatant was absorbed onto a C18 column. The column was eluted step-wise with 10, 20, 30, 40, 50, and 60% acetonitrile (CH3CN) in 0.1% trifluoroacetic acid (TFA). The greatest PRF activity was recovered in the 30% CH3CN/0.1% TFA fraction and was further purified by ion-exchange chromatography on SP-Sephadex, followed by gel-filtration on Sephadex G-50. The Sephadex G-50 fractions with major PRF activity were finally purified by two cycles of reverse phase HPLC, yielding a single peak of PRF. Amino acid, as well as sequence analyses, indicated that the highly purified PRF was oxytocin. Authentic oxytocin showed the same chromatographic behavior and biological activity as those of the isolated peptide. In another experiment, desalted crude extracts of rat and porcine posterior pituitary tissues were directly chromatographed by reverse phase HPLC, and each fraction was assayed for PRF activity. Only two areas showed PRF activity; the largest activity coeluted with oxytocin and the smaller one co-eluted with vasopressin. The fractions which coeluted with oxytocin also showed oxytocin immunoreactivity, as examined by RIA. The results clearly indicated that the major PRF in these posterior pituitary extracts was oxytocin.

Isolation of a novel 38 residue-hypothalamic polypeptide which stimulates adenylate cyclase in pituitary cells

A novel neuropeptide which stimulates adenylate cyclase in rat anterior pituitary cell cultures was isolated from ovine hypothalamic tissues. Its amino acid sequence was revealed as: His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln-Met-Ala- Val-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu-Gly-Lys-Arg-Tyr-Lys-Gln-Arg-Val-Lys-Asn-Lys-NH 2. The N-terminal sequence [1–28] shows 68% homology with vasoactive intestinal polypeptide (VIP) but its adenylate cyclase stimulating activity was at least 1000 times greater than that of VIP. It increased release of growth hormone (GH), prolactin (PRL), corticotropin (ACTH) and luteinizing hormone (LH) from superfused rat pituitary cells at as small a dose as 10 −10M (GH, PRL, ACTH) or 10 −9M (LH). Whether these hypophysiotropic effects are the primary actions of the peptide or what physiological action in the pituitary is linked with the stimulation of adenylate cyclase by this peptide remains to be determined.

Interaction between vasoactive intestinal polypeptide (VIP) and peptide histidine isoleucine (PHI) in stimulating the secretion of prolactin from rat anterior pituitary cells in vitro

Interaction between vasoactive intestinal polypeptide (VIP) and peptide histidine isoleucine (PHI) in regulating the secretion of prolactin (PRL) from the pituitary was investigated in the rat in vitro using two different methods: (1) short incubation of anterior pituitary cells and (2) superfusion of the pituitary cell column. PRL levels in the incubation medium were raised by addition of either VIP or PHI in concentrations of 10 −9 M to 10 −7 M in a dose-related manner. When both peptides were simultaneously added, additive stimulating effect on PRL release was obtained below the VIP concentration of 10 −7 M, in which no additive effect of PHI was revealed. PRL release from superfused rat pituitary cells was stimulated by 5-min pulses of VIP (10 −7 M), PHI (10 −7 M) and TRH (10 −8 M). Infusion of VIP for 200 min in the concentration of 0.3 × 10 −7 M resulted in an increase in basal release of PRL and blunted PRL release induced by not only VIP but also PHI stimulation, whereas PRL release induced by TRH was not affected. Infusion of PHI (0.3 × 10 −7 M, 0.7 × 10 −7 M and 10 −7 M) for 200 min also dose-relatedly suppressed PRL release induced by not only PHI but also VIP without any change in PRL release induced by TRH. These findings suggest that VIP and PHI may act through a common binding site on the pituitary lactotroph in the rat.

Contrôle de la libération de la GH par le GRF et la prostaglandine E2 dans un système de périfusion de cellules anté-hypophysaires de rat

Using superfused rat pituitary cells we showed that the growth hormone-releasing factor (GRF) and prostaglandin E2 (PGE2) increased growth hormone (GH) secretion in a similar manner in terms of kinetics and amplitude of responses. Moreover, our data suggest that they stimulate GH secretion mainly by cAMP-dependent mechanisms.