Milk-ejecting Activity Research Articles

Relatively greater increase of oxytocin than vasopressin in the supraoptic nucleus of rats after hypophysectomy.

The vasopressor and milk ejection activities were estimated in the supraoptic region of the hypothalamus of rats which had been hypophysectomized 30 min, 4 and 8 days earlier. Both pressor and milk ejection activities were significantly greater 4 days after hypophysectomy than those in sham-operated control rats. Eight days after hypophysectomy, pressor activity decreased below control values but milk ejection activity was still significantly raised. Increased biological activity after hypophysectomy is thus associated with the previously observed increase in neurosecretory material within the supraoptic nucleus so these results provide additional evidence that neurosecretory material represents stored hormone. Milk ejection activity increased relatively more than pressor activity which supports the suggestion that the final stage in the formation of neurohypophysial hormones occurs as they pass from the hypothalamus to the neural lobe. If this final maturation normally occurs more slowly for oxytocin than for vasopressin, the obstruction to the flow of hormones down the neural stalk caused by hypophysectomy would result in a greater increase in milk ejection than pressor activity.

Isolation and some observations of the properties of a bovine neurohypophysial milk-ejecting factor.

A substance possessing milk-ejecting activity has been isolated from an acetone powder preparation of bovine posterior pituitary glands by Sephadex G-25 chromatography of the neurophysin-neurohypophysial hormone complex. While the material possessed an oxytocic activity of 2.8 IU/mg as measured on the isolated rat uterus, the milk-ejecting activity was more than three fold greater, 9.6 IU/mg. The peptide had an antidiuretic activity of 0.133 IU/mg and a pressor activity of 0.083 IU/mg. Neither the uterine-stimulating action nor the pressor activity was destroyed by incubating the peptide with 0.01 M sodium thioglycollate at 65 degrees C for 5 min. The oxytocic activity was antagonized neither by 1.4 X 10(-6) M atropine nor 3.3 X 10(-7) M phenoxybenzamine.

Synthesis and some biological properties of 1-deamino-4-glu-oxytocin (1-beta-mercaptopropionic acid-4-glutamic acid-oxytocin) and its use in preparing a hormone-agarose complex.

1-Deamino-4-glu-oxytocin (1-beta-mercaptopropionic acid-4-glutamic acid - oxytocin) was synthesized by sequential reduction by sodium in liquid ammonia and oxidation by hydrogen peroxide of the octapeptide derivative, S-benzyl-beta-mercaptopropionyl-tyrosyl-isoleucyl-gamma-O-benzyl-glutamyl-asparaginyl-S-benzyl-cysteinyl-prolyl-leucyl-glycinamide. The oxidation analogue was isolated and purified by partition chromatography in two different solvent systems followed by exclusion chromatography on Sephadex G-25. It was found to possess approximately 13 I.U. of uterotonic activity, 34 I.U. of milk ejection activity, and 83 I.U. of milk ejection-like activity per milligram, measured on an isolated strip of lactating mouse mammary gland. 1-Deamino-4-Glu-oxytocin was coupled to AH-Sepharose 4B by the way of the free gamma-carboxyl group of its residue of glutamic acid. The water soluble 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride caused the coupling with approximately 70% effectiveness. The resultant peptide-agarose complex had low biological potency in the assay of milk ejection-like activity.

STUDIES ON PHARMACOLOGICAL AND BIOCHEMICAL PROPERTIES OF DEAMINO-DICARBA-[GLY7]-OXYTOCIN (Y-5350)

AbstractPharmacological and biochemical properties were investigated on de-amino-dicarba-[Gly7]-oxytocin (Y-5350). This is a newly developed compound, in which the disulfide bond and [Pro7] of deamino-oxytocin are substituted by an ethylene linkage and glycine respectively. Bioassayed Y-5350 exhibited 202.6 u/mg of oxytocic activity, 4.6 u/mg of avian depressor activity, 441.2 u/mg of rat milk-ejecting activity and 0.02 u/mg of rat antidiuretic activity, however, a depressor activity was also evident in rats. In particular, the duration of antidiuretic activity was short. Moreover, the oxytocic activity in pregnant rats and rabbits was weak in comparison with oxytocin. Cumulative dose-response studies were carried out on the isolated rat uterus using van Dyke-Hasting solution. The intrinsic activity was the same level as that of oxytocin, and the pD2 value was 8.62. Oxytocic activity was much enhanced by the existence of 0.5 or 2 mM magnesium ion in vitro.However, the agreement between in vivoand in vitrooxytocic activity was not complete when assay was carried out in the presence of 2 mM magnesium ion. Oxytocin was inactivated by the serum of pregnant women. In the experiment using rats, oxytocin was also destroyed by the extracts of uterus, kidney and liver. In contrast, Y-5350 was not destroyed by any of the enzyme solutions mentioned above.

The effect in vitro of prostaglandins with oxytocin on the milk-ejection activity of lactating sow mammary tissue.

A study by Turker & Kiran (1969) revealed that single retrograde intraarterial (i.a.) injections of prostaglandin E1 (PGE1) did not produce a change in milk-ejection pressure in the lactating rabbit, but could inhibit the milk-ejection response to oxytocin. The minimal dose range of PGE1 required to produce an inhibition of milk-ejection pressure by oxytocin was 0·1 to 1 μg PGE1 i.a. Halder, Maiweg & Grosvenor (1970) showed that PGE1 could abolish the milk-ejection activity of oxytocin in the rat. A study by McNeilly & Fox (1971) indicated that PGE1, PGE2, PGF1α and PGF2α, all possessed inherent milk-ejection activity in the guinea-pig. They found that in no case did the i.a. injection of any of these four prostaglandins in amounts up to 30 μg cause any decrease

Milk-ejecting activity induced by prostaglandin F2α

Prostaglandins have been shown to have physiologic effects upon several organs of the reproductive system. An outstanding example is the effect upon the uterine smooth muscle. Although there is some contradicting evidence about its action upon the mammary myoepithelium in animals, no similar action has been described on the human myoepithelium. This study describes the milk-ejecting activity of prostaglandin F2α (PGF2α) in lactating women. Evidence is presented suggesting that this activity is mediated through the central release of oxytocin. Preliminary in vitro results obtained in the rat suggest the possibility of an additional peripheral effect of PGF2α upon the myoepithelium.

Characterization of a Proposed Oxytocin Receptor in Rat Mammary Gland

Abstract [3H]Oxytocin, incubated with mammary tissue from the lactating rat, was bound specifically to three particulate fractions obtained by homogenization and centrifugation at 1,000, 20,000, and 105,000 x g. Radioactivity in the cytosol appeared to be unbound oxytocin, as determined by gel filtration on Sephadex G-100. Oxytocin was distributed about equally among the three particulate fractions and could not be correlated with either the amount of protein or the enzymes putatively characteristic of cell membranes, mitochondria, or microsomes. [3H]Oxytocin, added directly to particles sedimenting between 1,000 and 20,000 x g, was bound with an apparent Kd of 7.6 x 10-10 m under optimal conditions; 0.28 pmole of oxytocin was bound per mg of protein. Binding was maximal in 1 hour at 20°. The affinities of synthetic analogues of oxytocin, [4-threonine]oxytocin g oxytocin g [8-valine]oxytocin g [8-lysine]vasopressin g [4-proline]oxytocin, paralleled their milk-ejecting activities in rat mammary tissue. [3H]Oxytocin did not appear to be metabolized during incubation. Binding was enhanced by Mn2+ and Co2+ g Mg2+ g Zn2+ and was absent in the presence of 1 mm ethylenediaminetetraacetate. Ca2+, 50 mm, did not affect binding nor did 100 mm NaCl, LiCl, or KCl. Binding in 50 mm Tris-maleate buffer, containing 20 mm Mn2+, was optimum at pH 7.6. Trypsin depressed binding about 30%; phospholipases A, C, and D and neuraminidase did not. Sulfhydryl reagents such as N-ethylmaleimide and iodoacetate did not inhibit binding whereas p-hydroxymercuribenzoate did. Butanedione and sodium fluoride did not affect binding. Binding was markedly depressed by 1 mm ATP but not by guanosine triphosphate, cytosine triphosphate, hypoxanthine triphosphate, or uridine triphosphate. The binding material in the 20,000 x g particles of mammary tissue presumably is part of the oxytocin-receptor system because binding specificity paralleled biological specificity, and the high affinity of the binding sites for oxytocin is commensurate with physiological levels of the hormone. The receptor must be a protein, at least in part, because of its susceptibility to trypsin and p-hydroxymercuribenzoate.

Influence of Large Doses of Oxytocin on Milk Ejection and Metabolic Rate of Rat Mammary Gland

Milk ejection activity and milk synthesis were studied in primiparous lactating Wistar rats (9 to 12 days postpartum) 24h after a single intraperitoneal injection of 10 IU of oxytocin or 1ml saline. In a first series of experiments litters were allowed to suckle during this period. Gain in litter weight was blocked completely in the group treated with oxytocin with a decrease in oxygen consumption of mammary gland as compared with saline controls. However, lactose, glucose, and total sugar content did not differ between groups. In a second series of experiments pups were taken away immediately after injections. Weaning 24h decreased oxygen consumption of mammary gland in saline controls. Weaning together with injection of oxytocin caused an even more pronounced decrease. Lactose content of the weaned control group was lower than in the suckled control group and decreased after injection of oxytocin. In vitro milk ejection of small pieces of mammary gland also was completely inhibited in the oxytocin group. Prolactin administration to the oxytocin group increased both lactose and total sugar content of the gland. A pronounced increase in in vitro milk ejection activity in the oxytocin group followed prolactin treatment. Milk ejection in vivo, as measured by intra-mammary pressure, also was drastically inhibited by injection of oxytocin. In contrast to the in vitro experiments prolactin injections did not remove this inhibition, though lactose concentrations of mammary gland increased.

The effect of hypothalamic deafferentation upon the ultrastructure and hormone content of the paraventricular nucleus.

SUMMARY The ultrastructure and hormone content of the paraventricular nucleus was studied in female rats after surgical isolation of the hypothalamus (by preparation of hypothalamic islands) from the rest of the brain for 48 h. When compared with control animals no quantitative changes were observed in the proportion of cytoplasm occupied by endoplasmic reticulum, Golgi apparatus and mitochondria or in the number of lysosomes/100 μm2 of cytoplasm. However, there was a very significant (P < 0·001) increase in the number of neurosecretory granules observed in the perikarya of paraventricular cells in the deafferentated preparations. Bioassay of hypothalamic extracts for milk ejection and antidiuretic activities showed that there was a significant increase (P < 0·05) of the former in hypothalamic islands. The increase was greatest in the paraventricular region (P < 0·01). The hypothalamic content of antidiuretic activity was not changed significantly by deafferentation. The results demonstrate that the amount of milk-ejection activity in the paraventricular nucleus is related to the number of neurosecretory granules and raises the intriguing question of why deafferentation of the hypothalamus influences its milk-ejection activity more than its antidiuretic activity.

The distribution of vasopressin and oxytocin in the hypothalamoneurohypophysial system of the guinea-pig.

1. The ratio of the content of vasopressin to that of oxytocin (V/O ratio) was estimated in the supraoptic nucleus (SON), paraventricular nucleus (PVN) and posterior pituitary gland (PIT) of guinea-pigs.2. Extracts were assayed for antidiuretic activity to estimate vasopressin and for milk-ejecting activity to estimate oxytocin. In assays for milk-ejecting activity, trypsin was used to inactivate vasopressin in the extracts.3. The mean V/O ratios in the SON, PVN and PIT were 28, 8.5 and 7.0 respectively in male guinea-pigs, 6.8, 7.4 and 6.9 in non-lactating females, and 5.1, 3.3 and 6.6 in lactating females.4. The distribution of the hormones within the hypothalamus is discussed in relation to their independent release in response to electrical stimulation of the SON and PVN.

Effect of different doses of ethanol on the milk-ejecting reflex in lactating women

Milk-ejecting activity was determined in lactating women 2 to 8 days after parturition in response to suckling of 10 minutes' duration by means of intraductal pressure recordings. The magnitude of the milk-ejecting response was estimated by measuring planimetrically the area under the pressure tracings during each suckling period. Recordings were obtained during normal conditions and after administration of different doses of ethanol. Considerable variation in the magnitude of the milk-ejecting response was observed under these conditions. Ethanol in doses up to 1 Gm. per kilogram did not significantly reduce the response, but after administration of 1 to 2 Gm. per kilogram of ethanol a significant reduction of the milk-ejecting response was observed. This inhibition appeared to be dose dependent. There was no difference in the response of the mammary gland to injected oxytocin under normal conditions and under the influence of ethanol, indicating that the inhibition of the milk-ejecting response was not peripheral.

Lactation following therapeutic abortion with prostaglandin F 2 .

THERE is little information concerning the effects of the prostaglandins on the mammary gland. Pregnant rats lactate after premature parturition induced by prostaglandin F2α (PGF2α)1, and lactogenesis is induced through the release of prolactin2. It has also been shown that PGE1 decreases the milk-ejection activity of oxytocin in the rabbit and rat3,4 whilst in the guinea-pig5 the milk-ejection activity of oxytocin is not decreased by PGE1, PGE2, PGE1α or PGF2α, which in this species all possess milk-ejection activity per se.

Low oxytocin secretion in diabetes insipidus associated with normal labor

A 35-year-old woman with diabetes insipidus of 23 years' duration was studied by recording uterine and milk-ejecting activities. Observed mammary gland hypersensitivity to exogenous oxytocin suggests that this woman had low levels of endogenous oxytocin but was able to function normally because of end-organ adaptation and hyperreactivity to oxytocin. Despite this, normal labor occurred. Uterine activity was in the low normal range, but the uterus was not hyperreactive to exogenous oxytocin. This suggests that other factors were producing a normal labor in this patient.

Milk-Ejection Activity in Goat Plasma during Parturition

SUMMARY Milk-ejection activity was not detected in goat plasma during the last 6 weeks of pregnancy. There were measurable levels during the first stage of labour, which increased during the second stage of labour to a maximum coincident with greatest distension of the vagina. Thereafter milk-ejection activity fell after delivery. These findings support the view that oxytocin plays a physiological role both in the initiation and expedition of parturition.

Excretion of oxytocin and vasopressin in human urine.

ABSTRACT A method has been developed for the estimation of oxytocin in human urine. The assay of oxytocin was based on the milk-ejecting activity measured in lactating rats following a preceding chromatographic concentration of oxytocin. Recovery experiments of oxytocin were performed, and the yield was about 70%. No endogenous milk-ejecting activity was detected, i. e. the excretion of endogenous oxytocin, if any, was less than 2 mU per 24 h. The 4 hour urine excretion of vasopressin and oxytocin was respectively about 5% and 0.2% of the subcutaneously injected dose.

Identification of neurohypophysial hormones with their antisera.

Identifying posterior pituitary hormones in body fluids or neurohypophysial extracts was heretofore partially achieved by using pharmacologic potency ratios or semispecific inactivation by thioglycolate or enzymes. Production of antisera against oxytocin and lysine-vasopressin has prompted us to test their specificity against lysine-vasopressin, arginine-vasopressin, arginine-vasotocin, and oxytocin. In ethanol anesthetized rats, antidiuretic and milk-ejection activities were assayed for each peptide-antiserum combination after 0, 30, 60, and 90 min of incubation. Results indicate that (a) oxytocin antiserum inactivates oxytocin, but not arginine-vasopressin, lysine-vasopressin, or arginine-vasotocin; vasopressin antiserum inactivates arginine-vasopressin and lysine-vasopressin, but neither oxytocin nor arginine-vasotocin; (b) an identifiable antigenic site exists for each hormone; (c) relatively specific identifications of natural neurohypophysial peptides are possible using antisera and bioassays; (d) this method is promising for identifying neurohypophysial peptides in body fluids and pituitary extracts; and (e) active and passive immunization against oxytocin and vasopressin may increase our understanding of their physiologic functions.

Failure of 1-L-penicillamine-oxytocin and 1-deaminopenicillamine-oxytocin to inhibit the uterine effect of oxytocin in rabbits in vivo.

1-l-Penicillamine-oxytocin and 1- deaminopenicillamine-oxytocin are analogues of oxytocin that have been found to be potent inhibitors of the oxytocic activity of oxytocin in the rat. The 2 analogues were tested on the rabbit uterus in vivo with regard to the uterine response both to endogenously released oxytocin and to intravenously administered synthetic oxytocin. They were found to be devoid of inhibitory activity when administered in molar ratios of inhibitor to oxytocin between 170:1 and 10000:1. Both analogues had an oxytocic effect on the rabbit uterus in vivo but were approximately 500 to 1000 times less potent than oxytocin in the same preparation. Neither analogue inhibited the milk-ejection effect of oxytocin as judged by the milk yield obtained by the pups from unanesthetized mothers. Both analogues had milkejecting activity estimated on the basis of the milk yield in anesthetized rabbits. This activity was about 50 to 100 times less than that of synthetic oxytocin. A sensitive chronic prepar...

Interaction of prostaglandin E 1 with oxytocin on mammary gland of the lactating rabbit

Prostaglandin E 1, when given by close arterial or intravenous injection did not produce any change in the milk-ejection pressure of the lactating rabbit but it decreased the milk-ejection activity of oxytocin. Adrenergic blockade did not influence this inhibition. It is concluded that the inhibitory action of prostaglandin E 1 on oxytocin-induced milk-ejection activity of the lactating rabbit is a consequence of a direct action of the lipid on myoepithelial cells.

Milk-ejecting and antidiuretic activities under neurohypophyseal inhibition with alcohol and water overload

Antidiuretic activity was measured by determining free water clearance and urinary osmolality, controlled by glomerular filtration rate (Ccr) and solute excretion (Cosm). Milk-ejecting activity was assayed by recording intraductal mammary pressures. Baby's suckling was used as a stimulus to evoke milk ejection. Water overload (20 ml. per kilogram of body weight) and ethyl alcohol were used as inhibitors of the neurohypophyseal function. In subjects with water overload or blood alcohol concentrations between 50 and 100 mg. per 100 ml., inhibition of the antidiuretic activity without modifications of the milk ejection was observed. These results demonstrate that a differential inhibition of the gland was obtained, reinforcing our previous observations suggesting different neural pathways and/or mechanisms, controlling the release of oxytocin and antidiuretic hormone. On the contrary, blood alcohol concentrations over 200 mg. per 100 ml. were able to completely inhibit both activities, demonstrating that high doses of alcohol cannot be used to produce specific inhibition of oxytocin release.

Milk-ejection activity (oxytocin) in peripheral venous blood in man during lactation and in association with coitus.

The direct estimation of oxytocin in blood during suckling in women has only been reported by two groups of workers, who obtained conflicting results. Hawker & Robertson (1957) and Hawker, Walmsley, Roberts, Blackshaw & Downes (1961) failed to detect a change in the oxytocin content of peripheral venous blood during suckling. However, Coch, Fielitz, Brovetto, Cabot, Coda & Fraga (1968) demonstrated the presence of oxytocin (12–25 μu./ml. plasma) in internal jugular venous blood collected from women during suckling. The present study investigates the possible release of oxytocin during suckling and coitus in man by direct estimation of the hormone in the blood. No previous estimation during human coitus has been reported, but in domestic animals an increase in the oxytocin content of the blood has been claimed after mating (Walmsley, 1963) and after artificial stimulation of the genitalia (Fitzpatrick, 1957; Roberts & Share, 1968). Three suckling experiments, with the same