Hans H. Zingg

Inhibition of oxytocin receptor function by direct binding of progesterone

The steroid hormone progesterone (P4) is essential for establishing and maintaining pregnancy in mammals. One of its functions includes maintenance of uterine quiescence by decreasing uterine sensitivity to the uterotonic peptide hormone oxytocin. Although it is generally held that steroid hormones such as P4 act at a genomic level by binding to nuclear receptors and modulating the expression of specific target genes, we show here that the effect of P4 on uterine sensitivity to oxytocin involves direct, non-genomic action of P4 on the uterine oxytocin receptor (OTR), a member of the G-protein-coupled receptor family. P4 inhibits oxytocin binding to OTR-containing membranes in vitro, binds with high affinity to recombinant rat OTR expressed in CHO cells, and suppresses oxytocin-induced inositol phosphate production and calcium mobilization. These effects are highly steroid- and receptor-specific, because binding and signalling functions of the closely related human OTR are not affected by P4 itself but by the P4 metabolite 5β-dihydroprogesterone. Our findings provide the first evidence for a direct interaction between a steroid hormone and a G-protein-coupled receptor and define a new level of crosstalk between the peptide- and steroid-hormone signalling pathways.

The oxytocin receptor

Novel sites of oxytocin receptor expression have recently been detected, including breast cancer cells, bone cells, myoblasts, cardiomyocytes and endothelial cells. These discoveries have greatly expanded the possible spectrum of oxytocin action beyond its classic role as an inducer of uterine contractions and milk ejection. Additional advances in the understanding of oxytocin receptor structure-function relationships, receptor trafficking and novel receptor-linked signaling cascades have made this receptor an attractive model for the study of G-protein-linked receptor function. Finally, the tocolytic efficiency of the oxytocin receptor antagonist atosiban, recently approved for clinical use in Europe, has opened new avenues for the prevention and treatment of preterm labor.

Oxytocin: Crossing the Bridge between Basic Science and Pharmacotherapy

Is oxytocin the hormone of happiness? Probably not. However, this small nine amino acid peptide is involved in a wide variety of physiological and pathological functions such as sexual activity, penile erection, ejaculation, pregnancy, uterus contraction, milk ejection, maternal behavior, osteoporosis, diabetes, cancer, social bonding, and stress, which makes oxytocin and its receptor potential candidates as targets for drug therapy. In this review, we address the issues of drug design and specificity and focus our discussion on recent findings on oxytocin and its heterotrimeric G protein‐coupled receptor OTR. In this regard, we will highlight the following topics: (i) the role of oxytocin in behavior and affectivity, (ii) the relationship between oxytocin and stress with emphasis on the hypothalamo–pituitary–adrenal axis, (iii) the involvement of oxytocin in pain regulation and nociception, (iv) the specific action mechanisms of oxytocin on intracellular Ca2+ in the hypothalamo neurohypophysial system (HNS) cell bodies, (v) newly generated transgenic rats tagged by a visible fluorescent protein to study the physiology of vasopressin and oxytocin, and (vi) the action of the neurohypophysial hormone outside the central nervous system, including the myometrium, heart and peripheral nervous system. As a short nine amino acid peptide, closely related to its partner peptide vasopressin, oxytocin appears to be ideal for the design of agonists and antagonists of its receptor. In addition, not only the hormone itself and its binding to OTR, but also its synthesis, storage and release can be endogenously and exogenously regulated to counteract pathophysiological states. Understanding the fundamental physiopharmacology of the effects of oxytocin is an important and necessary approach for developing a potential pharmacotherapy.

Oxytocin and vasopressin gene expression during gestation and lactation

Levels of rat hypothalamic oxytocin (OT) and vasopressin (AVP) mRNA were determined during gestation and lactation using a densitometric hybridization assay. Pregnancy induced a gradual rise in OT mRNA, reaching, by day 18, a level exceeding control by a factor of 2.5. Throughout the lactation period OT mRNA remained elevated at levels corresponding to 3 times that of control. Surprisingly, the dynamics of AVP mRNA accumulation paralleled closely the profile observed for OT mRNA throughout the time period studied. We conclude that in late pregnancy and lactation the expression of both, the OT and the AVP gene is stimulated in parallel by mechanisms operating at a pretranslational level, involving increased gene transcription or mRNA stabilization, or both. Further characterization of the two mRNA species revealed that both mRNAs are endowed with very long poly(A) tails of greater than 200 nucleotides, under conditions of both, normal and stimulated states of gene expression. The role, if any, of the prolonged poly(A) tails in mRNA stability remains to be determined.

Oxytocin and oxytocin receptor gene expression in the uterus.

Publisher Summary The posterior pituitary contains a strong uterotonic activity known as the nonapeptide oxytocin (OT). It has been determined that uterus itself represents a major site of OT production. Therefore, the activation of uterine contractions by circulating OT is only one aspect of OT action. A significant portion of OT activity originates from within the uterus and, therfore, acts via autocrine and paracrine mechanisms. This chapter provides an overview of the role of circulating oxytocin and its receptors. The uterus contains its own intrinsic OT system. The ligand and its cognate receptor are both expressed in the same organ and, to some extent, in the same cells. The experiments described in the chapter showed that the genes for the receptor and its cognate ligand were subject to strict and very dramatic regulation during gestation and also, to a lesser extent, during the estrous cycle.

Ontogeny of hypothalamic vasopressin, oxytocin and somatostatin gene expression

The expression of 3 neuropeptide genes, vasopressin (AVP), oxytocin (OT) and somatostatin (SOM), was studied in the developing rat hypothalamus using Northern blot analysis combined with densitometric scanning. A unique profile of developmental expression was established for each of the 3 genes. SOM mRNA is detectable at embryonic day 14 and reaches 40% of the adult levels by embryonic day 18. By contrast, accumulation of AVP and OT mRNA is mainly a postnatal event. AVP mRNA, although detectable in the late embryo, rises gradually after birth and attains 40% of adult levels after the second postnatal week. Maturation of OT gene expression occurs even later and parallels AVP gene expression with a lag time of one week. Observed increases in mRNA levels are due to an upregulation of gene expression since they occur essentially following cessation of neuronal cell proliferation. The rise in AVP and OT mRNA accumulation coincides with the establishment of synaptic input to AVP and OT neurons. Expression of the SOM gene, by contrast, occurs prior to neuronal cell differentiation and points to a possible function of SOM in the embryonic brain.

REGULATION OF COX-2 GENE EXPRESSION IN RAT UTERUS IN VIVO AND IN VITRO

Prostaglandins are involved in mediating several important processes in mammalian reproduction, including the initiation of parturition. In the present study, we examined the expression in the rat uterus of two-rate limiting enzymes involved in prostaglandin production, cyclooxygenase (COX) 1 and 2. Expression of the COX-2 gene in the pregnant rat uterus gave rise to a single mRNA transcript of approximately 4.4 kb. COX-2 mRNA levels increased 3.5 fold between day 7 of pregnancy and the onset of parturition on day 22. In contrast, COX-1 mRNA levels remained constant during the same period. To investigate factors involved in mediating the regulation of COX-1 and COX-2 gene expression, rat endometrial stromal and epithelial cell lines, were used. In the stroma-derived cell line, CUS-V2, COX-2 gene expression was demonstrated by reverse transcriptase/polymerase chain reaction (RT-PCR) and by immunocytochemistry. In these cells, COX-2 gene expression was inducible by the cytokines interleukin-1 beta and tumor necrosis factor alpha, but not by interleukin-6. The two former cytokines also induced prostaglandin F2 alpha production. In contrast, COX-1 gene expression was constitutive in this cell line. In the endometrial epithelium-derived cell line, CUE-P both COX-1 and COX-2 genes were expressed in a constitutive fashion. In conclusion, the present in vivo and in vitro data indicate that decidual COX-2, but not COX-1, gene expression is regulated during pregnancy and implicate specific cytokines as possible inducers within the decidua.

A Novel Biased Allosteric Compound Inhibitor of Parturition Selectively Impedes the Prostaglandin F2α-mediated Rho/ROCK Signaling Pathway

The prostaglandin F2α (PGF2α) receptor (FP) is a key regulator of parturition and a target for pharmacological management of preterm labor. However, an incomplete understanding of signaling pathways regulating myometrial contraction hinders the development of improved therapeutics. Here we used a peptidomimetic inhibitor of parturition in mice, PDC113.824, whose structure was based on the NH2-terminal region of the second extracellular loop of FP receptor, to gain mechanistic insight underlying FP receptor-mediated cell responses in the context of parturition. We show that PDC113.824 not only delayed normal parturition in mice but also that it inhibited both PGF2α- and lipopolysaccharide-induced preterm labor. PDC113.824 inhibited PGF2α-mediated, Gα12-dependent activation of the Rho/ROCK signaling pathways, actin remodeling, and contraction of human myometrial cells likely by acting as a non-competitive, allosteric modulator of PGF2α binding. In contrast to its negative allosteric modulating effects on Rho/ROCK signaling, PDC113.824 acted as a positive allosteric modulator on PGF2α-mediated protein kinase C and ERK1/2 signaling. This bias in receptor-dependent signaling was explained by an increase in FP receptor coupling to Gαq, at the expense of coupling to Gα12. Our findings regarding the allosteric and biased nature of PDC113.824 offer new mechanistic insights into FP receptor signaling relevant to parturition and suggest novel therapeutic opportunities for the development of new tocolytic drugs.

Biosynthesis of immunoreactive somatostatin by hypothalamic neurons in culture.

The neuronal biosynthesis of somatostatin-like immunoreactivity (SLI) was investigated using mechanically dispersed neonatal rat hypothalamic cells kept in culture for up to 6 wk. Immunohistochemically, SLI was specifically localized to a small subpopulation of parvicellular neurons and their cell processes. By radioimmunoassay the cellular SLI content declined steadily during the first 2 wk in culture (nadir value of 60 fmol/dish at day 15) but then increased progressively to reach a maximum value of 381 fmol/dish at day 46. Gel chromatographic analysis showed this immunoreactivity to consist of forms corresponding to tetradecapeptide somatostatin (S-14), somatostatin-28 (S-28), and a 15,000-mol-wt molecule. After incubation of the cells with [3H]phenylalanine, the cellular extracts, purified by adsorption to C18 silica, contained material that bound specifically to an immobilized antisomatostatin antibody. Analysis by gel chromatography and high performance liquid chromatography of the specifically bound label provided evidence for the presence of labeled S-14, S-28, and the 15,000-mol-wt molecule. Pulse-chase experiments (20-min pulse, 20-min chase) demonstrated a transfer of radioactivity from the 15,000-mol-wt form to material corresponding to S-14 as well as to S-28. These studies demonstrate that cultured hypothalamic neurons are capable of synthesizing three somatostatin-like peptides (15,000-mol-wt SLI, S-28, S-14), one of which (15,000-mol-wt SLI) serve as a biosynthetic precursor for both S-28 and S-14. This in vitro system should provide a powerful tool for further investigation of the biosynthesis and regulation of biosynthesis of somatostatin in the hypothalamus.

Somatostatin precursors: Evidence for presence in and release from rat median eminence and neurohypophysis

Abstract Characterization of somatostatin-like immunoreactivity in rat median eminence and neurohypophysis by gel chromatography yielded two high molecular weight forms in addition to the tetradecapeptide somatostatin. The two larger molecules comprised 5% and 35% of the total tissue immunoreactivity, showed molecular weights of 25000 and 4000 dalton and were both releaseable in vitro in response to depolarizing stimuli. Their characteristic elution volumes remained unchanged after treatment with dithiothreitol or boiling in 8 M urea. Gentle trypsinization of the 25000 dalton molecule resulted in partial conversion into immunoreactive material coeluting with the tetradecapeptide somatostatin. Since the antibody employed in these studies is specific for the central and C-terminal portions of the tetradecapeptide somatostatin the present data suggest that both high molecular weight forms represent N-terminal extensions of somatostatin and that the 25000 molecular weight material might represent a prohormone for somatostatin.