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 epsilon-amino group of lysine in position 4 or 8 or, in the case of oxytocin, through the N-terminal amino group of 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.     

Magnesium and biological activity of oxytocin analogues modified on aromatic ring of amino acid in position 2.

For the purpose of evaluating substitution effects in the ortho, meta or para positions of the aromatic ring of tyrosine or phenylalanine in position 2 of oxytocin on uterotonic activity in vitro in the presence and absence of magnesium ions, six new analogues of oxytocin ([D- and L-m-methylphenylalanine2]oxytocin, [D- and L-m-methoxyphenylalanine2]oxytocin and [D- and L-o-methyltyrosine2]-oxytocin) were synthesized and several previously described analogues resynthesized. For the phenylalanine series, it is found that, in the absence of magnesium ions, substitution of the ortho and meta positions leads to loss of intrinsic activity (the analogues are antagonists) in contrast to the para position. In the tyrosine series, only methyl substitution in the meta position has this effect (substitution of ortho position only attenuates the agonistic biological activity). Addition of Mg ions restores to a certain degree the agonistic activity in the case of the o-methylphenylalanine analogue and enhances the agonistic activity of o-methyltyrosine oxytocin. All other analogues keep the original qualities as in the absence of Mg. Molecular modelling calculations of the structure of the above analogues was carried out to help explain these findings of the molecular level.     

Antipyrogenic properties of oxytocin

Effects of oxytocin on pyrogenal or endogenous pyrogen-induced fever were studied. Intramuscular injection of oxytocin (0.2 micrograms/kg every half an hour) did not significantly affect the pattern of pyrogenal-induced fever. Constant intravenous injection of oxytocin (0.4 and 4 micrograms/kg/h) 2-4.5 h after pyrogenal decreased the rectal temperature, on an average, by 24% and 31%, respectively. Endogenous pyrogen fever was not attenuated by intravenous oxytocin (4 micrograms/kg/h). The antipyrogenic effect of oxytocin is related to inhibition of endogenous pyrogen synthesis rather than to blockade of its action, which is indicated by a decreased second peak of the temperature curve, inhibition of endogenous pyrogen synthesis in vitro, and persistence of the hyperthermic effect of endogenous pyrogen.     

Desaminopenicillamine tocinoic acid derivatives--inhibitors of oxytocin

Tocinoic acid analogs with penicillamine in place of one or both of the cysteine residues have been studied and [1-beta-mercaptopropionic acid, 6-penicillamine] tocinoic acid (dPen6TA) and [1-beta,beta-dimethyl-beta-mercaptopropionic acid, 6-penicillamine] tocinoic acid (dPen1Pen6TA) have been synthesized in solution. Biological activities of these 2 compounds and those of the previously synthesized [1-beta,beta-dimethyl-beta-mercaptopropionic acid] tocinoic acid (dPen1TA) have been assayed. It was found that dPen1TA and dPen1Pen6TA, both of which have a beta,beta-dimethyl-beta-mercaptopropionic acid in position 1, are strong inhibitors of the uterine activity of oxytocin in vitro (without Mg2+) with pA2 values of 7.1 and 7.8, respectively, whereas dPen6TA with penicillamine in position 6 is a mild agonist.     

Effect of oxytocin on concentration of PGF2 alpha in the uterine lumen and subsequent endometrial responsiveness to oxytocin in pigs

The aim of this study was to determine the effect of oxytocin on PGF2 alpha secretion into the uterine lumen of pigs and subsequent endometrial responsiveness to oxytocin in vitro. Cyclic, pregnant and oestradiol-induced pseudopregnant gilts were injected i.v. with vehicle or 20 iu oxytocin 10 min before hysterectomy on day 16 after oestrus. Concentrations of PGF2 alpha and 13,14-dihydro-15-keto PGF2 alpha (PGFM) were significantly increased in uterine flushings collected at hysterectomy (P < 0.05) in pregnant oxytocin-injected gilts. Concentrations of PGF2 alpha and PGFM were greater (P < 0.001) in pregnant than in pseudopregnant and cyclic gilts, and greater (P < 0.01) in pseudopregnant than in cyclic gilts. The ratio of PGFM:PGF2 alpha tended to be greater in cyclic (P < 0.06) and pseudopregnant gilts (P < 0.1) than in pregnant gilts. At 85 +/- 5 min after oxytocin injection, endometrium from each gilt was incubated for 3 h for determination of phosphoinositide hydrolysis and PGF2 alpha secretion in response to treatment with 0 or 100 nmol oxytocin l-1. Endometrial phosphoinositide hydrolysis in response to 100 nmol oxytocin l-1 in vitro was greater (P < 0.05) in cyclic oxytocin-injected gilts than in cyclic vehicle-injected gilts. Treatment with oxytocin in vitro did not stimulate phosphoinositide hydrolysis significantly in vehicle- or oxytocin-injected pregnant gilts or pseudopregnant gilts. Endometrial PGF2 alpha secretion increased after treatment with 100 nmol oxytocin l-1 in vitro in cyclic vehicle-injected (P < 0.01), cyclic oxytocin-injected (P < 0.01), pregnant vehicle-injected (P = 0.06), pseudopregnant vehicle-injected (P < 0.05) and pseudopregnant oxytocin-injected (P < 0.05) gilts, but not in pregnant oxytocin-injected gilts. The increase in PGF2 alpha in pseudopregnant oxytocin-injected gilts was less (P < 0.05) than that in cyclic oxytocin-injected gilts. These results indicate that oxytocin increases the concentration of PGF2 alpha and PGFM in the uterine lumen during pregnancy and may upregulate endometrial responsiveness to oxytocin during late dioestrus in pigs, but does not have the latter effect during early pregnancy or oestradiol-induced pseudopregnancy.     

Secretion of oxytocin and progesterone by ovine corpora lutea in vitro

The mechanisms involved in the control of oxytocin and progesterone secretion by the ovine corpus luteum have been investigated in vitro using luteal slice incubations. Oxytocin and progesterone were secreted at constant rates from luteal slices for 2 h of incubation (366 +/- 60 pg X mg X h and 18.9 +/- 0.18 ng X mg X h, respectively). Secretion of progesterone, but not of oxytocin, was significantly (p less than 0.02) stimulated in the presence of ovine luteinizing hormone. Incubation of luteal slices in medium containing 100 mM potassium, however, resulted in increased secretion of oxytocin and, to a lesser extent, of progesterone (294 +/- 59% and 142 +/- 15%, respectively, p less than 0.05). Basal oxytocin secretion was reduced during incubation in calcium-free medium, compared to secretion in the presence of calcium (70 +/- 15 and 175 +/- 25 pg X mg X 20 min, respectively, p less than 0.01), whereas progesterone secretion was not altered in the absence of calcium. Secretion of both hormones by luteal slices was stimulated by the addition of the calcium ionophore A23187 (p less than 0.05). Addition of prostaglandin F2 alpha (2.8 microM) had no effect on secretion of either oxytocin or progesterone. We have demonstrated that oxytocin and progesterone can be stimulated, independently, from corpus luteum slices incubated in vitro. The pattern of release is consistent with the proposal that oxytocin, but not progesterone, is associated with and actively released from luteal secretory granules. Our results also indicated that prostaglandin F2 alpha does not directly stimulate release of oxytocin or progesterone from luteal cells in vitro.     

Effect of oxytocin on the calcium pump in myometrium sarcolemma

Oxytocin (10(-7) M) administered inside the myometrium sarcolemma vesicles closed outward by the cytoplasmic side is shown to inhibit Mg2+, ATP-dependent Ca2+ accumulation in these structures having no effect on the passive release of cation out of them. According to these results and to the data available in literature on the inhibitory action of the peptide hormone on Mg2+, Ca2+-ATPase of myometrium sarcolemma a conclusion is drawn that oxytocin inhibits the Ca pump activity in plasma membranes of the myometrium cells.     

Greatly elevated and sustained secretion of oxytocin by bovine granulosa cells in serum-free culture

Bovine granulosa cells were cultured in collagen (Vitrogen 100)-pretreated wells using defined medium to study the secretion of oxytocin and progesterone under serum-free conditions. Secretion of oxytocin began spontaneously after the first day and was maintained at a high level during a 1 week culture period. Addition of serum to the medium reduced oxytocin concentrations by up to 90%. There were positive exponential relationships between oxytocin and progesterone concentrations and the inoculated cell density (range, 1.67 to 23.4 X 10(5) cells/ml/well). The results indicate that neither serum nor gonadotrophins are required for in vitro differentiation of bovine granulosa cells and that addition of serum may attenuate subsequent hormone secretion. This culture system should provide a better in vitro model for the study of ovarian oxytocin secretion than those previously described.     

Effects of oxytocin outside pregnancy

For a long time, oxytocin was regarded as a pregnancy hormone released by the hypophysis to stimulate labour and milk ejection. In the present survey, data have been collected from the literature to show the spectrum of the hitherto known functions of oxytocin outside pregnancy. It is now known that oxytocin receptors can occur almost ubiquitously in the organism, that oxytocin is also formed outside of the brain and that oxytocin has functions in a number of organs. In the first part of the survey, stimuli that contribute to an increase in oxytocin release are compiled. In the second part, details are given on the individual oxytocin targets. Although the majority of findings are based on the results of animal experiments, there are already a number of studies that indicate similar effects of oxytocin in humans. According to the current state of knowledge, oxytocin appears to be involved in functions in the following organs: male and non-pregnant female reproductive tract, pancreas, cardiovascular system, kidney, brain and breast. There are indications that oxytocin may also have actions in other organs. There continues to be a considerable need for research into oxytocin in order to better understand the physiological and pathophysiological actions and to be able to derive possible therapeutic uses. Further light on the spectrum of functions of oxytocin may be cast by the possibility of the use of oxytocin antagonists.     

Synthesis and some pharmacological properties of [Glu(OMe)4]oxytocin and [Mpr1, Glu(OMe)4]oxytocin.

[Glu(OMe)4]oxytocin (XVI) and [Mpr1, Glu(OMe)4]oxytocin (XVII) bearing a methyl ester group in place of the carboxamide group in position 4 of oxytocin were synthesized by (3 + 6) segment condensation using the S-trityl group for the protection of the cysteine side chains. Analogue XVI exhibited 10.5 U/mg in vitro uterotonic, and 42 U/mg avian vasodepressor, activity, and analogue XVII 21.4 U/mg and 82 U/mg of the respective activities. Both compounds showed no response in the rat pressor assay.     

NO inhibits supraoptic oxytocin and vasopressin neurons via activation of GABAergic synaptic inputs

To study modulatory actions of nitric oxide (NO) on GABAergic synaptic activity in hypothalamic magnocellular neurons in the supraoptic nucleus (SON), in vitro and in vivo electrophysiological recordings were obtained from identified oxytocin and vasopressin neurons. Whole cell patch-clamp recordings were obtained in vitro from immunochemically identified oxytocin and vasopressin neurons. GABAergic synaptic activity was assessed in vitro by measuring GABA(A) miniature inhibitory postsynaptic currents (mIPSCs). The NO donor and precursor sodium nitroprusside (SNP) and L-arginine, respectively, increased the frequency and amplitude of GABA(A) mIPSCs in both cell types (P < or = 0.001). Retrodialysis of SNP (50 mM) onto the SON in vivo inhibited the activity of both neuronal types (P < or = 0.002), an effect that was reduced by retrodialysis of the GABA(A)-receptor antagonist bicuculline (2 mM, P < or = 0.001). Neurons activated by intravenous infusion of 2 M NaCl were still strongly inhibited by SNP. These results suggest that NO inhibition of neuronal excitability in oxytocin and vasopressin neurons involves pre- and postsynaptic potentiation of GABAergic synaptic activity in the SON.     

Pharmacological chaperones for the oxytocin receptor increase oxytocin responsiveness in myometrial cells

Oxytocin is a potent uterotonic agent administered to nearly all patients during childbirth in the United States. Inadequate oxytocin response can necessitate Cesarean delivery or lead to uterine atony and postpartum hemorrhage. Thus, it may be clinically useful to identify patients at risk for poor oxytocin response and develop strategies to sensitize the uterus to oxytocin. Previously, we showed that the V281M variant in the oxytocin receptor (OXTR) gene impairs OXTR trafficking to the cell surface, leading to a decreased oxytocin response in cells. Here, we sought to identify pharmacological chaperones that increased oxytocin response in cells expressing WT or V281M OXTR. We screened nine small-molecule agonists and antagonists of the oxytocin/vasopressin receptor family and identified two, SR49059 and L371,257, that restored both OXTR trafficking and oxytocin response in HEK293T cells transfected with V281M OXTR. In hTERT-immortalized human myometrial cells, which endogenously express WT OXTR, treatment with SR49059 and L371,257 increased the amount of OXTR on the cell surface by two- to fourfold. Furthermore, SR49059 and L371,257 increased the endogenous oxytocin response in hTERT-immortalized human myometrial cells by 35% and induced robust oxytocin responses in primary myometrial cells obtained from patients at the time of Cesarean section. If future studies demonstrate that these pharmacological chaperones or related compounds function similarly in vivo, we propose that they could potentially be used to enhance clinical response to oxytocin.     

Conformationally restricted analogs of oxytocin; stabilization of inhibitory conformation

Analogs of oxytocin containing tetrahydroisoquinoline carboxylic acid (Tic) of L or D configuration in position 2 were synthesized and their biological activities were tested. Both analogs showed negligible agonist activity in uterotonic, galactogogic, and pressor assays, but they are in vitro uterotonic inhibitors. In comparison with oxytocin analogs containing L- or D-phenylalanine in position 2, the analog with the D-configuration of the conformationally fixed aromatic residue has significantly increased inhibitory activity which suggests that the proper conformation for the interaction with the receptor, but not for its activation, was stabilized. 1H NMR and CD studies, supported by theoretical calculations, suggest that the conformational properties of the analog containing D-tetrahydroisoquinoline carboxylic acid are similar to those of [2-D-phenylalanine]oxytocin.     

Enhanced secretion of oxytocin from bovine granulosa cells treated with adrenal steroids

Bovine granulosa cells were exposed in vitro to various adrenal steroids (cortisol, cortisone, corticosterone, aldosterone; 1 mumol/l), in the presence and absence of stimulation by ascorbic acid (0.5 mmol/l), to determine the possible effects of these hormones on ovarian oxytocin and progesterone secretion. Only cortisol produced a consistent stimulation of the cells; the response was dose-related over the range 0.01 to 1.0 mumol/l and was greatly enhanced in the presence of ascorbate. The secretion of oxytocin was stimulated to a greater extent and with more consistency than was that of progesterone. Although the secretion of oxytocin could be stimulated by cortisol on the day of treatment, the cells also showed a delayed and persistent response to exposure earlier in the culture. It is concluded that cortisol may directly stimulate the secretion of ovarian oxytocin in the cow and that granulosa cells may respond in such a way as to smooth out the effects of short-term fluctuations in cortisol concentration.     

Enzyme immunoassay for oxytocin

A competitive, double antibody enzyme immunoassay for oxytocin in a heterologous system was developed. Horseradish peroxidase was conjugated with oxytocin using N-succinimidyl 3-(2-pyridyldithio) propionate, and rabbit anti-oxytocin serum was produced by immunization of oxytocin-bovine serum albumin complex which was prepared by the carbodiimide method. The sensitivity of the assay was 4 microIU/tube, which corresponded to 10 microIU per ml using 400 microliters of the sample which was extracted from the same volume of plasma by means of SEP-PAK C18 cartridges. The coefficients of variation for different levels of oxytocin ranged from 6.8-15.9% and 8.5-16.7%, for intra- and inter-assay. Recovery of oxytocin added to plasma after extraction was 99-117%. No or little cross-reaction with arginine- and lysine-vasopressin was found. Plasma oxytocin concentrations determined by the proposed enzyme immunoassay were well correlated with those determined by radioimmunoassay (r = 0.90).     

Uterine responsiveness of oxytocin and prostaglandin F2 alpha in heat-acclimated rats

1. Contractility, in vitro, was examined in uterine horns of rats acclimated to 35 degrees C and controls (22 degrees C). 2. Responses to oxytocin and prostaglandin F2 alpha were measured in the four stages of the estrus cycle and on day 4 of pregnancy. 3. Responses to oxytocin of uteri from heat acclimated rats were significantly depressed in estrus, metestrus and diestrus, while responses to prostaglandin F2 alpha were decreased in estrus and metestrus. 4. Responses to oxytocin and prostaglandin were slightly but insignificantly decreased in uteri from pregnant day 4 heat-acclimated rats.     

Continuous infusion of oxytocin prevents induction of uterine oxytocin receptor and blocks luteal regression in cyclic ewes

Continuous intravenous infusion of oxytocin (3 micrograms/h) between Days 13 and 21 after oestrus delayed return to oestrus by 7 days (length of cycle 23.3 +/- 0.6 days compared to 16.6 +/- 0.2 days in control ewes). At a lower infusion rate (0.3 micrograms/h) oxytocin delayed luteolysis in only 2 of 5 ewes. Treatment from Day 14, when luteolysis had already begun, was ineffective. Delay of luteal regression by oxytocin had no effect on the length of subsequent cycles. Measurement of circulating progesterone concentrations and luteal weight showed that prolongation of the oestrous cycle was due to prevention of luteal regression. Luteal regression and behavioural oestrus were induced during continuous oxytocin administration begun on Day 13 when cloprostenol was given on Day 15 (mean cycle length, 17.3 +/- 0.21 days). Continuous oxytocin infusion from Day 13 blocked the rise in uterine oxytocin receptor concentrations which normally precedes oestrus. Mean receptor concentrations in caruncular and intercaruncular endometrium and in myometrium were 76, 36 and 9 fmol/mg protein on Day 17 in ewes receiving continuous oxytocin (3 micrograms/h); in control ewes these values were 675, 638 and 130 fmol/mg protein respectively at oestrus. Receptor concentrations on the day of oestrus in ewes receiving oxytocin and cloprostenol were not significantly different from those in control ewes (649, 852, and 109 fmol/mg protein respectively). Since cloprostenol, a PGF-2 alpha analogue, overcame the antiluteolytic action of oxytocin, it is suggested that continuous oxytocin treatment may inhibit uterine production of PGF-2 alpha, possibly by down regulating the uterine oxytocin receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Non-peptide oxytocin agonists

A library of compounds targeted to the vasopressin/oxytocin family of receptors was screened for activity at a cloned human oxytocin receptor using a reporter gene assay. Potency and selectivity were optimised to afford compound 39, EC50 = 33 nM. This series of compounds represents the first disclosed, non-peptide, low molecular weight agonists of the hormone oxytocin (OT).