Milk-ejecting Activity Research Articles

Ability to Keep Milk-Ejecting Activity after Premature Birth

The article examines the modern data on the importance of breast milk in nursing premature babies. It is shown that the amount of breast milk in women, who gave birth prematurely, decreases rapidly, especially when it is impossible to get a baby latched on to the breast. There is a negative correlation between gestational age and duration of lactation. According to the opinion of both doctors and the majority of mothers, pumping out breast milk and using it in feeding a premature baby is an important psychological and physiological factor linking a mother and a child at the intensive care unit. Individual breast milk banks are a new safe and effective method for long-term keeping of breast milk.

The influence of exogenous oxytoxin on labelling of secretory epithelium of mouse mammary gland by [3H]leucine, evidenced by autoradiography.

It has been suggested that oxytocin, besides its milk-ejecting activity, is also involved in the hormonal regulation of the mammary gland secretory cells. The available data, however, are conflicting. In this study two independent experiments, separated by a certain time interval show that oxytocin intravenously administered to mice at days 10-14 of lactation diminished the incorporation of [3H]leucine into the mammary gland tissue by 32 and 53 per cent, respectively. The neurohormone was co-injected with the tracer amino acid. The radioactivity taken up by the secretory cells was estimated by light microscopic autoradiography. The autoradiograms were evaluated by visual silver grain counting. Tissue radioactivity was measured by liquid scintillation counting. A milk stasis in the mammary gland induced by depriving the mice of suckling two hours before tracer injection had no influence on the secretory activity of the glandular cells. It is assumed that oxytocin has a direct effect on the milk-producing cells, and that the reduction in measurable radioactivity caused by the neurohormone may be due to an accelerated intracellular passage of labelled milk proteins.

Oxytocin in the human--regulation of derivations and destinations.

Those of us who study physiology are tempted to hope that the experimental perturbations which we induce affect only the organ which has ensnared our interest. This review uses physiological systems which incorporate oxytocin as models to indicate some of the ways in which departure from such investigative paradise occurs, and also to discuss some of the implications of a physiology which uses one compound in several distinct roles. The dual activities of oxytocin, on the uterus and on the mammary gland, for some time constituted the complete portfolio of the peptide’s functions in biology (1). However, now no longer is consideration of oxytocin confined to its two traditional roles. Investigations of oxytocin have demonstrated that oxytocin participates in a wide variety of activities in dispersed regions of the body, and is an element of the physiology of both sexes. Oxytocin is involved with sex, pain and moods, and its sites of action include the thymus, the ovary, and the pancreas. After the oxytocic activity and the milk ejecting activity of the posterior pituitary gland were recognised, a nonapeptide was found to be responsible for the effects. The structure of the compound was established (2, 3) and oxytocin was the first peptide hormone to be purified and synthesised, and available for pharmacologically defined studies. Modern molecular biology has provided information of the oxytocin system (Fig. 1). The oxytocin gene has been mapped to 20p13 (4) although the nature of the regulatory elements critical to cell-specific expression of the oxytocin gene are still ill-defined (5). The molecular structure of the oxytocin receptor has been characterised (6) and the gene has been localised to 3p25–3p26 (7–9). It is assumed that oxytocin has evolved by duplication of an ancestor gene which oxytocin and vasopressin have in common. The vasopressin/oxytocin prohormone ancestor was present in Archaemetazoa, from which vertebrates and invertebrates diverged 600 million years ago (10, 11). This review is designed first to serve as a reminder that a compound can affect multiple entities, secondly to consider some of the processes which control interaction between physiological units, and thirdly to point to the multifaceted effects, including alterations of behaviour, a pathological change can have. A variety of control mechanisms which are incorporated into oxytocin systems are summarised in Table 1 and corresponding examples indicated in the text by upper case letters (A–O). This review considers effects induced by oxytocin which have been observed in human studies, which after all is the ultimate target of most projects, with only occasional reference to antecedent animal investigations. Although animal studies have suggested several activities for oxytocin which are not discussed in this review it is necessary to note that there are some functions of oxytocin which are species specific. Examples are the apparently opposite effects of oxytocin on adrenocorticotrophin (ACTH) in humans (in which oxytocin is reported to be inhibitory) and in rats (in which oxytocin is stimulatory); another is the differences in the temporal profile of oxytocin in the cerebrospinal fluid (CSF) of humans and rats; a third is the distribution of oxytocin binding sites in the brain.

Mortyn Jones Memorial Lecture. Limbic regions mediating central actions of oxytocin on the milk-ejection reflex in the rat.

Central oxytocin administration has a profound facilitatory effect on the patterning of the milk-ejection reflex in the lactating rat. Lesion and microinjection studies indicate that this action is, in part, mediated via a population of limbic neurones in the bed nuclei of the stria terminalis and ventrolateral septum, which have been shown to possess oxytocin receptors and to be activated by selective oxytocin-receptor agonists in vitro. In vivo electrophysiological recordings reveal that some of these neurones display cyclical activity which is highly correlated to each milk ejection, and are rapidly activated following i.c.v. administration of oxytocin, coincident with the facilitation of milk ejection activity. A hypothetical model is proposed in which this population of limbic neurones serves to gate the activity of a pacemaker which, in turn, coordinates the bursting of hypothalamic magnocellular neurones. The oxytocin innervation of these neurones and their expression of oxytocin receptors increases in the postpartum period, and the resultant enhanced sensitivity leads to a greater facilitatory response during lactation. Inhibitory opioid and noradrenergic inputs which converge on these oxytocin-sensitive neurones may function to switch off the facilitatory circuit during periods of stress. Thus, this population of limbic neurones participates in the regulation of neuroendocrine activity during lactation by providing an appropriate degree of feedback to alter the patterning of the milk-ejection reflex.

Characteristics of the spontaneous milk ejecting activity occurring during human lactation.

Continuous recordings of intramammary pressure performed in 158 normal lactating women, and 22 women during spontaneous labor were retrospectively analyzed in order to characterize spontaneous milk ejecting activity (SA). SA appeared as spurts of contraction waves as early as the first post-partum day and throughout the first two months of lactation. The spurts of SA differed from reflex milk ejection evoked by baby's suckling. It showed two different patterns: a) single contraction waves with a rapid rise time, a slow decay time, high amplitude and a short duration; b) multiple contraction waves with a longer rise and decay times, progressively decreasing intensity and a large duration depending on the number of contractions per spurt. The multiple pattern occurred in 34.2% (41/120) of patients at 2.7 +/- 0.1 days of post-partum and increased to 84.3% (59/70) at 34.5 +/- 1.2 days. Accordingly, duration and the area under contraction curves increased throughout lactation. Mammary gland sensitivity to exogenous oxytocin (OT) did not change during the 2 months studies. SA was significantly greater in full nursing mothers than in the partially nursing ones. The existence of a system for milk-ejection different from baby's suckling was further described. A neuroendocrine mechanism and the release of increasing amounts of OT are suggested as important factors in the control of this phenomenon.

Isolation and structure elucidation of bovine pineal arginine vasopressin: arginine vasotocin not identified

A large number of reports have demonstrated the presence of neurohypophysial hormone-like peptides in mammalian pineal glands and an antigonadotropic function has been ascribed to pineal arginine vasotocin (AVT). We have undertaken large scale purification of bovine pineal neurohypophysial hormone-like substances which demonstrate mouse mammary milk-ejection activity (ME-activity) in vitro. Peptides with ME-activity were extracted from more than 5 kg of bovine pineal glands. ME-activity containing peptides were found in both high (Mr approximately 10,000-15,000) and low (Mr approximately 500-1000) Mr species from Sephadex G-25 chromatography of 0.2 N acetic acid extracts. After ultrafiltration in 5% formic acid, the neurohypophysial hormone-like peptides were localized to an ultrafiltration Mr 500-1000 retentate. A homogeneous peptide, which shared an identical retention time (RT) and amino acid sequence with synthetic 8-arginine vasopressin (AVP), was isolated by serial semipreparative high performance liquid chromatography. On the other hand, the non-mammalian nonapeptide AVT was not identified.

Synthesis and biological activities of oxytocin and lysine vasopressin analogs containing glutamic acid gamma-hydrazide in position 4.

Solution methods, using N-hydroxysuccinimide esters, were used to synthesize [Glu(NHNH2)4] oxytocin and [Glu(NHNH2)4, Lys8] vasopressin. In these analogs of neurohypophyseal hormones, the side-chain carboxamide function of a glutamine residue is formally replaced by a hydrazide group at position 4. The hormone analogs were assayed for uterototonic activity, milk ejection activity, antidiuretic activity, and rat pressor activity. The specific biological activities of the oxytocin and vasopressin analogs were decreased compared to the respective parent hormones in all assay systems.

Fluorescent, photoaffinity, and biotinyl analogs of oxytocin

This study reports the solid phase synthesis and biological activities of two oxytocin analogs, [1-desamino, 4-lysine, 7-(L-3,4,-dehydroproline)]oxytocin and [1-desamino, 4-threonine, 7-(L-3,4-dehydroproline),8-lysine]oxytocin, and several fluorescent, photoaffinity, or biotinylated derivatives of these analogs and of oxytocin. The activities (in IU/mg) of the lysine-containing parent compounds, respectively, were as follows: uterus (without Mg ++) 4.8 and 54; uterus (with Mg ++) 19 and 440; milk ejection 65 and 414. The above analogs were coupled through the chemically reactive ϵ-amino group of lysine in position 4 or 8 or, in the case of oxytocin, through the N-terminal amino group to fluoresceine, photoaffinity, or biotinyl ligands. Fluoresceine coupled in position 1 of oxytocin gave an analog of low to moderate uterine (3.8 without Mg + and 1.9 with Mg ++) and milk ejection (7.9) activities. Analogs with biotin or fluoresceine coupled to lysine in position 4 had moderate uterine (11 and 23 without Mg ++; 38 and 11 with Mg ++) and milk ejection (33 and 13) activities. Analogs with fluoresceine, photoaffinity, or biotinyl labels coupled to lysine in position 8 retained good uterine (106, 62, and 147 without Mg ++; 79, 78, and 509 with Mg ++) and milk ejection (101, 181, and 247) activities and represent potentially useful experimental tools for studying hormone-receptor interactions and for receptor localization and isolation.

Arginine vasopressin and oxytocin in the bovine adrenal gland.

The concentrations of immunoreactive oxytocin and arginine vasopressin (AVP) and their respective neurophysins (NpI and NpII) were compared in bovine adrenal cortex and medulla. While the concentration of AVP was similar in both tissues there was more NpII in the medulla. The medulla also contained much more oxytocin and NpI than the cortex. The extracted AVP and oxytocin had identical retention times to those of the synthetic peptides on high-performance liquid chromatography (HPLC) and were biologically active in assays for antidiuretic and milk-ejection activity (with potencies of 310 units/mg and 340 units/mg respectively). Adrenal NpI and NpII behaved identically to commercially available neurohypophysial proteins on HPLC. Oxytocin, NpI and AVP were assayed in five subcellular fractions of bovine adrenal medulla prepared on discontinuous sucrose gradients. A high proportion of each co-localized with noradrenaline and adrenaline in the chromaffin granule fraction. Binding of [3H]AVP and [3H]oxytocin to crude bovine adrenal medulla membranes was dependent upon both time and temperature. The binding sites were specific and saturable: studies with the V1 AVP antagonist d(CH2)5Tyr(Me)AVP and the V2 agonist 1-deamino-8-D-AVP indicated that the AVP receptor was V1 in specificity. Scatchard plots showed that each ligand interacted with a single high-affinity, low-capacity binding site: oxytocin dissociation constant (Kd) 3.1 +/- 0.29 nmol/l, maximum binding capacity (Bmax) 89.6 +/- 18.4 fmol/mg protein (n = 3); AVP Kd 0.73 +/- 0.02 nmol/l, Bmax 26.5 +/- 8.3 fmol/mg protein (n = 3).(ABSTRACT TRUNCATED AT 250 WORDS)

ChemInform Abstract: SYNTHESIS AND SOME PHARMACOLOGICAL PROPERTIES OF (4‐THREONINE,7‐SARCOSINE)OXYTOCIN, A PEPTIDE WITH HIGH OXYTOCIC POTENCY, AND OF (4‐THREONINE,7‐N‐METHYLALANINE)OXYTOCIN

Two analogues of oxytocin, [Thr4,Sar7]- and [Thr4,MeAla7]oxytocin, were synthesized and their pharmacological properties investigated. [Thr4,Sar7]oxytocin was found to exhibit high biological activity (uterotonic activity of 1174 +/- 104 and milk ejection activity of 731 +/- 57 units/mg) and high selectivity for oxytocin-like relative to vasopressin-like activities (antidiuretic activity of 0.037 +/- 0.012 unit/mg, undetectable pressor activity). [Thr4,MeAla7]oxytocin was characterized by markedly lower biological activities. In both analogues, the additivity of the effects of the residues in positions 4 and 7 of oxytocin on the biological activity of the analogues was ascertained.

Synthesis and some pharmacological properties of [4-threonine,7-sarcosine]oxytocin, a peptide with high oxytocic potency, and of [4-threonine,7-N-methylalanine]oxytocin.

Two analogues of oxytocin, [Thr4,Sar7]- and [Thr4,MeAla7]oxytocin, were synthesized and their pharmacological properties investigated. [Thr4,Sar7]oxytocin was found to exhibit high biological activity (uterotonic activity of 1174 +/- 104 and milk ejection activity of 731 +/- 57 units/mg) and high selectivity for oxytocin-like relative to vasopressin-like activities (antidiuretic activity of 0.037 +/- 0.012 unit/mg, undetectable pressor activity). [Thr4,MeAla7]oxytocin was characterized by markedly lower biological activities. In both analogues, the additivity of the effects of the residues in positions 4 and 7 of oxytocin on the biological activity of the analogues was ascertained.

Synthesis and some pharmacological properties of oxytocin and vasopressin analogues with sarcosine or N-methyl-L-alanine in position 7.

Eight analogues of oxytocin and arginine-vasopressin were synthesized, in which the proline residue in position 7 was replaced by either sarcosine or N-methylalanine; some of the pharmacological properties of these analogues were evaluated. In peptides containing a beta-mercaptopropionic acid residue in position 1, the additivity of the effects of deletion of the amino group in position 1 and of the above-noted replacements in position 7 on biological properties of these analogues was ascertained. All of the analogues were found to be potent in either antidiuretic or uterine activity and also selective in action. From the point of view of pharmacological properties, substitution of sarcosine in position 7 of oxytocin gave analogues with higher oxytocic and milk-ejecting activities than did the substitution of N-methylalanine. The opposite structure-activity relationship was observed with arginine-vasopressin, where the N-methylalanine-containing analogues were more potent than the sarcosine-containing analogues with respect to pressor activity and also, if not deaminated, with respect to antidiuretic activity.

Biofunctional evaluation of a hydrogen bond stabilizing the conformation in the cyclic part of oxytocin.

[5-beta-Malamidic acid]oxytocin was synthesized to study the importance of the hydrogen bond between the C=O of Tyr2 and the peptide N-H of Asn5 for the stabilization of a biologically functional conformation of oxytocin. This analog lacks the peptide N-H at residue 5 required for the formation of a hydrogen bond with the C-O of Tyr2. [5-beta-Malamidic acid] oxytocin exhibited 45.1 +/- 2.5 U/mg and 65.6 +/- 5.9 U/mg of uterotonic activity, in vitro, in the absence and in the presence, respectively, of Mg2+, 147 +/- 14 U/mg of uterotonic activity in vivo, 203 +/- 13 U/mg of milk-ejecting activity, 0.37 +/- 0.03 U/mg of pressor activity and 0.32 +/- 0.29 U/mg of antidiuretic activity. It is concluded that devoid of the hydrogen bond under question, an oxytocin-like peptide can still assume the conformation needed to interact with the oxytocin receptors.

Synthesis, pharmacological, conformational, and dynamic studies of the potent hormone antagonists [1-penicillamine, 4-threonine]-oxytocin and [1-penicillamine, 2-phenylalanine, 4-threonine]-oxytocin. Conformational and dynamic considerations in the design of antagonists.

The solid phase synthesis of [1-penicillamine, 4-threonine]-oxytocin and [1-penicillamine, 2-phenylalanine, 4-threonine]-oxytocin is reported. The two compounds have no in vitro milk ejecting activity and no in vivo or in vitro oxytocic activity, but both are potent antagonists in these three assay systems. In the in vitro oxytocic assay, [1-penicillamine, 4-threonine]- and [1-penicillamine, 2-phenylalanine, 4-threonine]-oxytocin have pA2 values of 7.55 +/- 0.04 and 7.67 +/- 0.02, respectively, and both inhibit the uterine contractile response to oxytocin in nonpregnant and pregnant rats. [1-Penicillamine, 2-phenylalanine, 4-threonine]-oxytocin has a weak antipressor activity and at high doses, consistently caused a weak and transient fall in blood pressure in the rat. Carbon-13 nuclear magnetic resonance chemical shift parameters and spin-lattice relaxation times (T1) indicate that these two new oxytocin antagonists have very similar conformation and dynamic properties to oxytocin inhibitors which have previously been examined. These results are discussed in terms of conformational and dynamic models of oxytocin antagonism at the uterus. It is suggested that conformational restrictions at the 2- and 4-positions of penicillamine-1 analogues of oxytocin are important to antagonist activity and potency.

Biofunctional evaluation of a hydrogen bond stabilizing the beta-turn in the acyclic part of oxytocin.

In a continued effort to determine the importance of the hydrogen bonds for stabilization of the biologically active conformation of oxytocin, deamino-[9-glycolicamide] oxytocin was synthesized in order to study, in this respect, the hydrogen bond between the peptide N--H of Gly9 and the C=0 of Cys6. In this analog the amide linkage between residues at positions 8 and 9 is replaced by an ester. Thus the residue at position 9 cannot be involved in hydrogen bond formation with the C=O of Cys6. Deamino-[9-glycolicamide] oxytocin exhibited 134 +/- 13 U/mg and 355 +/- 48 U/mg of uterotonic activity in absence and in presence, respectively, of Mg2+, 108 +/- 8 U/mg of milk-ejecting activity, 0.35 +/- 0.03 U/mg of pressor activity and 2.5 +/- 0.1 U/mg of antidiuretic activity. It is concluded that the hydrogen bond under question is not critical for the conformation required for biofunctional interaction of oxytocin with its receptors in the uterus, mammary gland and other target organs.

Studies on the importance of the asparagine residue in oxytocin. Synthesis and some pharmacological properties of [1-alpha-mercaptoacetic acid, 5-isoasparagine] oxytocin.

[1-Alpha-Mercaptoacetic acid, 5-isoasparagine] oxytocin was synthesized to study the effects of moving the side chain carboxamide group of the amino acid residue in position 5 of oxytocin from the beta to the alpha position. The analog has an isoasparagine residue in position 5 and the 20-membered ring size of oxytocin is maintained by substituting cysteine in position 1 of oxytocin by alpha-mercaptoacetic acid. The analog was found to possess 0.098 +/- 0.002 U/mg of uterotonic activity but no milk-ejecting, antidiuretic or rat pressor activity could be detected. The substance did not inhibit the uterotonic or milk-ejecting activity induced by oxytocin nor the antidiuretic or rat pressor responses to the USP posterior pituitary standard. These results, together with the data available in the literature, indicate that an analog of oxytocin lacking the asparagine residue in position 5 is neither an agonist nor an antagonist. The observations may mean that the asparagine residue is critically important for the interaction of oxytocin with its receptor.

Inhibitory effect of adrenaline on oxytocin release in the ewe during the milk-ejection reflex

Milk-ejection activity was determined in the blood plasma of ewes during normal milking and during milking when adrenaline was injected intravenously before or after udder stimulation. It was found that administration of adrenaline either before or after udder washing, decreased the oxytocin concentration and milk yield but increased the yield by hand-stripping. Adrenaline also retards the average time for peak oxytocin concentration. These results and the use of a beta-receptor blocker to inhibit the effect of adrenaline at the myoepithelial cell level indicate that in ewes adrenaline can prevent the release of oxytocin from neurohypophysis.

Hormone Action: Oxytocin-Induced in vitro Milk Release

H ORMONE ACTION IS AN AREA of physiology that presents a challenge to instructors seeking economical procedures for demonstrating principles. The milk-ejecting activity of the neurohormone, oxytocin, on mammary gland tissue is a dynamic example of the mechanism of action of a hormone, yet it is simple and relatively inexpensive to demonstrate. The main physiological role of oxytocin, a hormone of the posterior pituitary gland of mammals (Linzell 1959), is to stimulate milk release from the mammary gland. Oxytocin is an example of a neurohormone because it is synthesized within neurons in the hypothalamus, an area of the lower part of the brain (diencephalon) responsible for the control of many physiological functions. Oxytocin is transported within these neurosecretory neurons to their neural endings in the posterior pituitary gland. The

Inhibitory effect of prostaglandin F2 alpha on oxytocin release and on milk ejection in lactating rats.

The effect of prostaglandin F2 alpha (PGF2alpha) on milk ejection and on oxytocin release during suckling for one or two periods of 30 min was studied in lactating rats. Doses of PGF2 alpha (20 or 40 phi g) were injected i.p. 15 min before the suckling period. Control rats were injected with physiological saline. An inhibition of milk ejection proportional to the dose of drug administered was obtained. A normal milk ejection response was induced with a small dose of oxytocin injected immediately before nursing to mothers treated with PGF2 alpha, indicating that the blocking effect was not due to a lack of mammary gland response. Two groups of mothers were injected with 40 phi g PGF2 alpha 2 and 4 h respectively before suckling. In both groups milk ejection was partially but significantly inhibited. In rats pre-treated with sodium pentobarbitone (3-5 mg/100 g body wt) to prevent the release of oxytocin induced by suckling, PGF2 alpha (10 or 20 phi g) did not modify the inhibition of milk ejection indicating that PGF2 alpha does not have milk-ejecting activity. The administration of oxytocin to anaesthetized rats, immediately before a second suckling period, induced a normal milk-ejection response while in the rats treated with PGF2 alpha, oxytocin was less effective. The results indicate that PGF2 alpha inhibited milk ejection by a central block on oxytocin release and that the lipid is not able to mimic peripherally the milk-ejecting activity of oxytocin.

Synthesis and some pharmacological properties of (2-tryptophan)-oxytocin.

Previous studies have suggested that an aromatic amino acid residue at position 2 in oxytocin facilitates the expression of the hormone's biolgocial activities. [2-Tryptophan]-oxytocin, in which a residue of tryptophan has replaced that of tyrosine in oxytocin, has been synthesized by the method of azide coupling of the N-terminal dipeptide and C-terminal heptapeptide amide. It was found to have approximately 0.1% of the potency of oxytocin in milk ejection and uterotonic biological activities.