Prolactin-inhibiting Activity Research Articles

Innovative formulations to enhance oral bioavailability of Agnus castus extract

The main characteristic constituents of Vitex agnus castus are diterpenes, iridoid glycosides, flavonoids, flavones and triglycerides. Therapeutic indications are premenstrual syndrome and menstrual cycle disorders [1]. In vitro and in vivo experiments have shown that Agnus castus extracts (ACEs) have dopaminergic, prolactin-inhibiting activity [2]. The project is aimed at developing new oral innovative formulations which can overcome the limited oral bioavailability of iridoid glycosides and flavonoids and thus increase the therapeutic efficacy. After optimization of a HPLC-DAD-QTOF analytical method for qualitative and quantitative characterization of consitutents of ACE, nanoemulsions were selected as lipid carriers. The preparations were performed by dissolving extract powder into the oil (triacetin)-surfactant (labrasol)-cosurfactant (Cremophor EL) mixture, adding water. The droplets in the microemulsion appeared dark, and the surroundings bright under Transmission Electron Microscopy with an average diameter of 11.15 ± 0.07nm. Polydispersity (PdI) was 0.07 ± 0.01 measured by a Dynamic Light Scattering. The aqueous solubility of the extract was improved: the extract at the concentration of 40 mg/mL is completely soluble in the nanoemulsion, while the solubility of extract in water resulted less than 6 mg. Finally passive intestinal absorption was evaluated using a Parallel Artificial Membrane Permeation Assay. The test evidenced a good increase of total constituents of the ACE permeated from donor to acceptor compartement.

Helix-loop-helix motif in GnRH associated peptide is critical for negative regulation of prolactin secretion

The GnRH associated prolactin inhibiting factor (GAP) reveals the signature sequence associated with the helix-loop-helix structural motif. A number of different peptide fragments of GAP were designed, synthesized and analysed by circular dichroism and by an in vivo assay for prolactin secretion inhibiting activity. Peptides corresponding to the two individual α-helices and a 44-residue peptide comprising the entire helix-loop-helix motif show significant helical propensity in circular dichroism spectra. However, a peptide corresponding to the loop sequence shows no helical propensity. Albeit, the peptide corresponding to helix-loop-helix motif was found to inhibit prolactin secretion and augment circulating levels of gonadotropins in the in vivo assay; other shorter peptides did not show such activity. The activity profile of the 44-residue peptide was biphasic and very similar to that of the recombinant GAP. Thus, the prolactin inhibiting activity of this factor is defined by its helix-loop-helix motif as in the case of the transcription factors of developmental genes. The structural features of a homology-based model of GAP in complex with E47, a ubiquitous HLH-type developmental gene regulator, are consistent with the structural requirements of the negative regulation of transcription by helix-loop-helix proteins.

Role of nitric oxide in control of prolactin release by the adenohypophysis.

Nitric oxide synthase-containing cells were visualized in the anterior pituitary gland by immunocytochemistry. Consequently, we began an evaluation of the possible role of NO in the control of anterior pituitary function. Prolactin is normally under inhibitory hypothalamic control, and in vitro the gland secretes large quantities of the hormone. When hemipituitaries were incubated for 30 min in the presence of sodium nitroprusside, a releaser of NO, prolactin release was inhibited. This suppression was completely blocked by the scavenger of NO, hemoglobin. Analogs of arginine, such as NG-monomethyl-L-arginine (NMMA, where NG is the terminal guanidino nitrogen) and nitroarginine methyl ester, inhibit NO synthase. Incubation of hemipituitaries with either of these compounds significantly increased prolactin release. Since in other tissues most of the actions of NO are mediated by activation of soluble guanylate cyclase with the formation of cyclic GMP, we evaluated the effects of cyclic GMP on prolactin release. Cyclic GMP (10 mM) produced an approximately 40% reduction in prolactin release. Prolactin release in vivo and in vitro can be stimulated by several peptides, which include vasoactive intestinal polypeptide and substance P. Consequently, we evaluated the possible role of NO in these stimulations by incubating the glands in the presence of either of these peptides alone or in combination with NMMA. In the case of vasoactive intestinal polypeptide, the significant stimulation of prolactin release was augmented by NMMA to give an additive effect. In the case of substance P, there was a smaller but significant release of prolactin that was not significantly augmented by NMMA. We conclude that NO has little effect on the stimulatory action of these two peptides on prolactin release. Dopamine (0.1 microM), an inhibitor of prolactin release, reduced prolactin release, and this inhibitory action was significantly blocked by either hemoglobin (20 micrograms/ml) or NMMA and was completely blocked by 1 mM nitroarginine methyl ester. Atrial natriuretic factor at 1 microM also reduced prolactin release, and its action was completely blocked by NMMA. In contrast to these results with prolactin, luteinizing hormone (LH) was measured in the same medium in which the effect of nitroprusside was tested on prolactin release, there was no effect of nitroprusside, hemoglobin, or the combination of nitroprusside and hemoglobin on luteinizing hormone release. Therefore, in contrast to its inhibitory action on prolactin release NO had no effect on luteinizing hormone release. Immunocytochemical studies by others have shown that NO synthase is present in the folliculostellate cells and also the gonadotrophs of the pituitary gland. We conclude that NO produced by either of these cell types may diffuse to the lactotropes, where it can inhibit prolactin release. NO appears to play little role in the prolactin-releasing action of vasoactive intestinal polypeptide and substance P, but mediates the prolactin-inhibiting activity of dopamine and atrial natriuretic factor.

Total solid-phase synthesis and prolactin-inhibiting activity of the gonadotropin-releasing hormone precursor protein and the gonadotropin-releasing hormone associated peptide.

The human gonadotropin-releasing hormone precursor protein, pHGnRH (Met-23-Ile69) (preproGnRH), and three of its fragment peptides, pHGnRH (Asp14-Ile69) (gonadotropin-releasing hormone associated peptide--GAP), pHGnRH (Phe38-Ile69), and pHGnRH (Ser47-Ile69), were assembled in a stepwise solid-phase cosynthesis employing Boc/Bzl tactics and an optimized acylation schedule which included recoupling steps with hexafluoro-2-propanol to help overcome the aggregation of the pendant peptide chains of the peptidoresin during difficult couplings. Reversed-phase high-performance liquid chromatography (HPLC) purification yielded products which were characterized by analytical reversed-phase HPLC, ion-exchange chromatography, capillary zone electrophoresis, SDS-polyacrylamide gel electrophoresis, and ion-spray mass spectrometry to reveal a high degree of homogeneity. Biological characterization demonstrated that only GAP stimulated luteinizing hormone and follicle-stimulating hormone release from primary cultures of rat anterior pituitary cells, while GAP, pHGnRH (Phe38-Ile69), and preproGnRH all inhibited prolactin release, with the latter being the most potent at concentrations comparable to bromocryptine. However, only GAP and pHGnRH (Phe38-Ile69) were able to displace a labeled gonadotropin-releasing hormone agonist from binding to rat pituitary membrane preparations. This first demonstration of significant biological activity with a precursor protein also suggests that the gonadotropin-releasing and prolactin release-inhibiting functions of GAP are not mediated through the same pituitary receptors.

Age-related release of prolactin by the pituitary and the pituitary-hypothalamic complex in vitro: an attempt to describe the development of the hypothalamic prolactin-inhibiting and -releasing activities in male rats

We have recently demonstrated that the pituitary hypothalamic complex (PHC) is a good model for studying interactions between the hypothalamus and pituitary in vitro. The amount of prolactin secreted by the PHC is an index of prolactin secreted by the pituitary in the presence of hypothalamic control, while the amount released by the whole pituitary alone is an index of prolactin secreted in the absence of hypothalamic control. The amount of prolactin secreted by the PHC has been regarded as an index of hypothalamic prolactin-releasing activity (HPRA), while the difference in the amounts of prolactin secreted by the whole pituitary and the PHC is the hypothalamic prolactin-inhibiting activity (HPIA). Attempts were made to correlate HPRA and HPIA to the development of serum concentrations of prolactin from days 7 to 77 in male rats. The HPRA increased steadily from days 7 to 56, decreased significantly on day 63 and thereafter remained unchanged until day 77. The HPIA was low on days 7 and 14 and increased steadily up to day 49, with no further significant variations. The developmental patterns of HPRA and HPIA were comparable up to day 49. Serum concentrations of prolactin increased significantly until day 28 and remained fairly constant until day 49. The weight of the pituitary gland increased from 1.0 +/- 0.03 mg (mean +/- S.E.M.) on day 7 to 7.76 +/- 0.32 mg on day 63 and remained unchanged thereafter. The weight of the hypothalamic islet was 31.5 +/- 2.88 mg on day 7, 34.83 +/- 1.45 mg on day 14 and 50.4 +/- 4.01 mg on day 21. After day 21 the weights of the hypothalamic islets were not significantly altered, except on day 49. It was concluded that serum concentrations of prolactin are regulated by interaction or competition between HPRA and HPIA at the level of the pituitary.

Orchidectomy induces temporal and regional changes in the synthesis and processing of the LHRH prohormone in the rat brain.

Recently, the sequence of the cDNA which encodes the LHRH-prohormone was elucidated from human placenta and human and rat hypothalamus and the corresponding amino acid sequence deduced (Seeburg et al., 1984 and Adelman et al., 1986). In addition to LHRH, the prohormone contains a 56 amino acid sequence, designated gonadotropin-releasing hormone associated peptide (GAP), which is attached to the C-terminus of the LHRH decapep-tide. Although not yet confirmed, the human GAP sequence has been reported to possess both gonadotropin-releasing and prolactin inhibiting activity (Nikolics et al., 1985). Additionally, a 13-amino acid fragment of the human GAP sequence (proLHRH 14–26) has been reported to stimulate gonadotropin release (Millar et al., 1986). Regardless of whether the non-LHRH portion of the LHRH prohormone contains biological activity, this sequence can serve as a valuable marker for studies of LHRH prohormone synthesis, processing and degradation. In order to initiate these types of studies, we have generated specific antisera (MC-1, 2 and 3) against a fragment of the human GAP sequence (proLHRH 38–66) and developed a radioimmunoassay procedure for the quantitation of GAP (Culler and Negro-Vilar, 1986). The antisera are specific for midportion sequences of the GAP molecule and do not cross-react with any other known brain peptide.

Purification of a non-dopaminergic and non-GABAergic prolactin release-inhibiting factor (PIF) in sheep stalk-median eminence.

Prolactin release-inhibiting factor (PIF) extracted from 1200 sheep stalk-median eminences was purified by gel filtration on a Sephadex G-25 column (4.5 X 150 cm). PIF activity was determined by measuring the inhibition of prolactin release from dispersed anterior pituitary cells of adult male or estrogen-primed, ovariectomized rats. Using this system, PIF was detected in tube fractions 122-127 (volume = 20 ml/tube). These fractions also contained LHRH and somatostatin; however, these peptides had no prolactin-inhibiting activity in the quantities present. No dopamine or gamma-aminobutyric acid (GABA) was detected in the active fractions by radioenzymatic assay and fluorophotoenzymatic assay, respectively. Furthermore, receptor blockers for dopamine or GABA did not interfere with the PIF activity. These findings indicate that the PIF activity cannot be attributed to either dopamine or GABA, both of which are known to inhibit prolactin release, and provide evidence for the presence of a non-dopaminergic and non-GABAergic PIF within the hypothalamus.

Potent in vivo and in vitro prolactin inhibiting activity of sauvagine, a frog skin peptide.

The effect of sauvagine (SAU), a frog skin peptide, on prolactin (PRL) levels was studied in vivo and in vitro. Subcutaneous administration of SAU (20 micrograms/kg) reduced plasma PRL levels in normal adult male rats and suppressed the suckling-induced rise of PRL in lactating rats even at doses of 1 and 5 micrograms/kg. Perfusion of isolated and dispersed rat pituitary cells in vitro with increasing doses of SAU (from 5 x 10(-10) to 1.7 x 10(-8)M) induced a significant dose-related decrease of PRL secretion in the eluate. These results indicate that SAU is a potent PRL inhibiting factor and that its action is exerted at the pituitary level. If SAU or a SAU-related peptide is present in the mammalian brain, it can be tentatively hypothesized that this peptide plays an important role in the control of PRL secretion.

Dopaminergic influences on prolactin synthesis and release from rat anterior pituitary cultures

Dopaminergic influences on prolactin release from lactotrophs have been studied using the rat anterior pituitary cell culture. The prolactin inhibiting activity of hypothalamic extracts was examined in relation to dopamine. Dopamine inhibited prolactin secretion from the rat anterior pituitary cell culture in a dose dependent fashion. The median effective dose was 2 x 10 -7 M and the maximal inhibition (70-90 ~o of the control value) was shown by 10 -5 M dopamine. Further increase in dopamine concentration did not result in any further inhibition of prolactin secretion. Dopamine antagonists such as haloperidol and (§ could inhibit the effect of dopamine on prolactin release. A Schild plot from the relationship between dopamine and (+)butaclamol in prolactin release in the anterior pituitary cell culture showed that pAz value of (+)butaclamol is 8.9 with a slope of 1.00. High concentrations of haloperidol or (+) butaclamol (both above 10-SM) caused a marked inhibition in prolactin release initially, followed by some irreversible cytotoxicity on the anterior pituitary cells. The major damage

ChemInform Abstract: ERGOT ALKALOIDS. SYNTHESIS OF NITROSOUREA DERIVATIVES OF ERGOLINES AS POTENTIAL ANTICANCER AGENTS

AbstractDas Amino‐methyl‐ergolin (I) reagiert mit dem Isocyanat (II) zum Harnstoff (III), der die Nitrosoderivate (IVa) und (IVb) ergibt.

Some 6-alkyl derivatives of D-8-ergolin-I-ylacetamide

D-6-Alkyl-8-cyanomethylergolines-I were converted either by known procedures via the corresponding methyl esters I-III and hydrazides IV-VI to amides VII-IX, or by hydration of the nitrile group under the conditions of ionic pair extraction to amides VIII and X-XIII. In the same system D-6-methyl-8-ergolin-I-ylacetamide (DeprenonR) was prepared from D-6-methyl-8-cyanomethylergoline-I. Amides VII, VIII and XI had a considerably higher prolactin inhibiting activity than Deprenon.

Some 6-substituted derivatives of D-8-cyanomethylergoline-I and D-8-methylergoline-I

Compounds III-XX exhibiting antilactation and antinidation effects in tests on rats were prepared on N(6)-alkylation of D-8-cyanomethylergoline-I (I) or D-8-methylergoline-I (II) with corresponding bromo (chloro) derivatives in dimethylformamide. The most distinct prolactin-inhibiting activity was found in compound III.

Ergot alkaloids. Synthesis of nitrosourea derivatives of ergolines as potential anticancer agents

Nitrosourea derivatives of ergolines have been synthesized for the purpose of obtaining agents with both prolactin-and tumor-inhibitory activity. Two derivatives of 8-amino-6-methylergoline (3), 8-[3-(2-chloroethyl)-3-nitrosoureido]-1-nitroso-6-methylergoline (5c) and 8-[3-2-chloroethyl)-3-nitrosoureido]-6-methylergoline (5a), have been prepared. In addition, nitroso (7) and chloroethylcarbamyl (8) derivatives of elymoclavine (6) are reported. Compounds 5a and 5c have activity against L1210 leukemia in mice but only moderate prolactin-inhibiting activity. The chloroethylcarbamyl derivative 8 of elymoclavine is a potent prolacting inhibitor.

Prolactin-inhibiting activity of gamma-aminobutyric acid-mimetic drugs in the male rat

Prolactin-inhibiting activity of gamma-aminobutyric acid-mimetic drugs in the male rat

Inhibition of prolactin secretion by ergolines.

Peptide-containing ergot alkaloids and synthetic ergoline derivatives were administered to reserpine-treated male rats in order to evaluate their prolactin-inhibiting properties. Each compound was administered ip at a standard 50 ¼g/kg dose. The 9,10-dihydrogenated ergots, dihydroergocornine, and dihydroergocryptine were able to inhibit prolactin to the same degree as ergocornine and ergocryptine. This is significant because the 9,10-dihydrogenation process removes the toxic vasoconstrictive properties from the molecule and allows the prolactin-inhibiting ability to remain intact. In addition, dose-response studies show that minute amounts of an ergot can cause a significant reduction in prolactin secretion. The failure of ergocorninine to inhibit prolactin secretion demonstrates that the stereochemical configuration of the molecule at the 8 position is important for prolactin-inhibiting ability. Interestingly, a number of simple ergot molecules lacking the peptide side chain are able to inhibit prolactin secretion. Substitution at the 1 or 2 position as well as hydrogenation of the indole reduces the prolactin-inhibiting activity. The most active compound is D6-methyl-8βergolineacetonitrile. The high prolactin-inhibiting potency of these compounds suggests that they are able to mimic prolactin-inhibiting factor (PIF). (Endocrinology94: 1171, 1974)

Effects of L-dopa on serum prolactin and PIF in intact and hypophysectomized, pituitary-grafted rats.

The effects of L-dopa were assessed on serum prolactin, on hypothalamic prolactininhibiting factor content and on serum prolactin releasing activity in hypophysectomized female rats with or without an anterior pituitary (AP) graft and in intact female rats. In 2 separate experiments a single ip injection of L-dopa in hypophysectomized rats with an AP graft decreased serum prolactin concentration by 50–60% within 2 hr. L-Dopa increased hypothalamic PIF content by 35% and the blood serum from these rats decreased pituitary prolactin release in vitro by 65%. In hypophysectomized rats with no AP transplant, L-dopa elicited similar changes in the ability of the hypothalamus and serum to influence prolactin release. In intact rats, injection of L-dopa depressed serum prolactin concentration, increased hypothalamic PIF content and evoked the presence of prolactin inhibiting activity in the serum. These results suggest that L-dopa inhibits pituitary prolactin release by increasing hypothalamic synthesis and relea...

Prolactin-inhibiting activity in hypophysial stalk blood and elevation by dopamine.

Des moities d'hypophyse incubees dans du plasma de la tige hypophysaire secretent moins de prolactine que les moities complementaires incubees dans du plasma de sang peripherique. Les glandes incubees dans du plasma de la tige hypophysaire de rats traites a la dopamine secretent moins de prolactine que celles incubees dans le plasma des rats temoins. L'activite inhibitrice de la prolactine dans le plasma de la tige hypophysaire peut Etre le resultat d'un facteur particulier, et la secretion de ce facteur serait gouvernee par un mecanisme «dopaminergique».

Effect of a hypothalamic extract on serum prolactin levels during the estrous cycle and lactation.

SummaryOn the morning of proestrus or estrus, mature rats were injected with an extract of eight rat hypothalami or an equivalent amount of cerebral tissue. Blood was removed just prior to injection of hypothalamic extract and at 1 and/or 4 hr after injection, for assay of prolactin by radioimmunoassay. In each of the three experiments, hypothalamic extract significantly depressed serum prolactin values, confirming the presence of prolactin-inhibiting activity in the hypothalamus. Cerebral extract had no effect. In another experiment, the litters of postpartum lactating rats were removed for 12 hr and the rats were injected with an extract of four or eight rat hypothalami or an equivalent amount of cerebral tissue. The litters were then returned for 3 hr of suckling. Blood samples taken before and after suckling, showed that neither brain extract prevented the suckling stimulus from producing a 5- to 6-fold increase in serum prolactin values. This indicates that 3 hr of suckling is a more powerful stimulu...

Studies on the purification of ovine prolcatin-inhibiting factor.

After several extractions, hypothalamic extract was subjected to gel filtration on long columns of Sephadex G-25. Prolactin-inhibiting activity of the fractions was estimated by their ability to block suckling-induced decline in pituitary prolactin. This inhibitory activity was recovered in each of 5 fractionations with Sephadex. In each instance the prolactininhibiting activity was found to be closely associated with luteinizing hormone (LH)-releasing activity as estimated by ovarian ascorbic acid depletion; however, in 3 of 5 experiments there was a tendency for the prolactin-inhibiting activity to be eluted from the column just prior to the emergence of the peak of the LH-releasing activity. When the zones with prolactin-inhibiting and LH-releasing activity from several columns were pooled, lyophilized and eluted from a carboxymethylcellulose column by application of ammonium acetate buffers of increasing pH and ionic strength, prolactin-inhibiting activity was again recovered from the column in fractions which also possessed LH-releasing activity. The peptide content in the highly purified prolactininhibiting factor (PIF) was estimated to be < 3 μg. Clearly, a manyfold purification of PIF was achieved without obtaining a clear-cut separation from the LH-releasing factor (LH-RF). The PIF was partially separated from follicle stimulating hormone-RF and removed from the zones which contained growth hormone-RF, melanocyte stimulating hormone-RF, thyrotropin-RF and vasopressin. (Endocrinology82: 1236, 1968)

Dose-response relationships between hypothalamic PIF and prolactin release by rat pituitary tissue in vitro.

A negative log-dose relationship was demonstrated when different amounts of rat hypothalamic extract containing prolactin-inhibiting activity (PIF) were incubated with rat anterior pituitary tissue, and the amounts of prolactin released were measured. Use of male rat pituitary glands in the incubation system proved to be more sensitive and showed less variability in response than when female ratpituitary tissue was incubated with hypothalamic extract. Rat cerebral extract at several dose levels had no significant effect on pituitary prolactin release in vitro. These results indicate that PIF exists as a specific hormone in the hypothalamus, and that the in vitro method is suitable for measuring changes in hypothalamic PIF content produced by different physiological states. {Endocrinology 80: 1170, 1967)