A López BernalMedical School, Division of Obstetrics and Gynaecology, Institute of Cellular Medicine, University of Newcastle, 3rd Floor, William Leech Building, Framlington Place, Newcastle upon Tyne NE2 4HH, UK
The state of contraction in smooth muscle cells of the human uterus is dependent on the interaction of activated forms of actin and myosin. Ras homology (RHO) proteins are small monomeric GTP-binding proteins that regulate actin polymerisation and myosin phosphorylation in smooth muscle cells. Their action is determined by their level of expression, GTP-bound state, intracellular localisation and phosphorylated status. Agonist activated RHO proteins bind to effector kinases such as RHO kinase (ROCK) and diaphanous proteins (DIAPH) to regulate smooth muscle contraction by two mechanisms: ROCK activates smooth muscle myosin either by direct phosphorylation at Ser19/Thr18 or through inhibition of myosin phosphatase which is a trimeric protein regulated by ROCK and by other protein kinases. Actin-polymerising proteins such as DIAPH homolog 1 increase filamentous actin assembly to enhance acto-myosin cross bridge formation and contraction. This review explores recent advances in RHO protein signalling in human myometrium and proposes areas of further research to investigate the involvement of these proteins in the regulation of uterine contractility in pregnancy and labour.
Prostaglandin F2α (PGF2α) has regulatory (mainly luteolytic) effects in the ovary but the mechanism of action is not completely understood. Reverse transcriptase–polymerase chain reaction (RT–PCR) techniques were used to demonstrate the presence of mRNA encoding the PGF2α receptor (FP receptor) in human granulosa–lutein cells. Specific primers for the amplification of cDNA were designed and yielded a single product of 696 bp corresponding to the FP receptor. The identity of this product was verified by sequencing. Fluprostenol, a selective FP receptor agonist, activated phospholipase C (PLC) and increased intracellular free calcium concentration, confirming the functional activation of the receptor. We have demonstrated by Western blotting that granulosa cells express PLC-β and PLC-γ isoforms. The cells responded to pervanadate with increased PLC activity and increased tyrosine phosphorylation, demonstrating a functional PLC-γ tyrosine kinase pathway. However, fluprostenol did not provoke any detectable tyrosine phosphorylation. Moreover, the effect of fluprostenol was inhibited through protein kinase C stimulation by phorbol 12,13-dibutyrate, and was not affected when cells were treated with phenylarsine oxide, which blocks tyrosine phosphorylation. These results suggest that the FP receptor activates PLC-β rather than PLC-γ isoforms. Fluprostenol-induced activation was pertussis toxin resistant. Granulosa cells express G proteins of the Gq family (resistant to pertussis toxin) and mRNA for both Gαq and Gα11 has been identified by RT–PCR. In conclusion, human granulosa cells have a functional FP receptor the effects of which are mediated through PLC-β activation probably via Gq/11.
Eukaryotic cells have an internal cytoskeletal scaffolding, giving them their distinctive shapes. The cytoskeleton enables cells to transport vesicles, undergo changes in shape, migrate and contract. This dynamic structure is formed by three classes of filamentous assembly: actin microfilaments, intermediate filaments and microtubules. In this investigation the cytoskeleton of cultured human myometrial cells was studied by immunohistochemistry using specific antibodies against vinculin, cytokeratin, vimentin, tubulin and RhoA, covalently labelled with a fluorescent tag. Polymerized actin was visualized with fluorescein-conjugated phalloidin. Myometrial cells were very rich in actin fibres, which generally appeared as parallel bundles along the longest axis of the cells. There was a strong expression of vinculin which concentrated at actin–vinculin focal adhesion sites. By contrast, intermediate filaments (vimentin and cytokeratin) were organized in a dense cytoplasmic meshwork which excluded the nuclear space. A similar pattern was observed for tubulin. RhoA had a diffuse distribution and was associated with actin fibres. Exposure of the cells to oxytocin provoked a 10% shortening of actin stress fibres. These results demonstrate that myometrial smooth muscle cells have a rich cytoskeletal structure and that agonists that stimulate myometrial activation provoke measurable changes in actin fibres which may be important for efficient contractility.
Oxytocin is used widely for the induction and augmentation of labour, but there is little information about the dynamics of oxytocin receptors in human myometrium during parturition, and the possible effect of oxytocin infusion. This information is important because G protein-coupled receptors, such as the oxytocin receptor, undergo desensitization after prolonged or repeated stimulation. The concentration of myometrial oxytocin receptors and the steady state of its mRNA were measured in patients undergoing Caesarean sections before or during spontaneous or induced labour. The concentration of receptors before labour was 477 (175-641) fmol mg(-1) protein (median, quartile range), and decreased to 140 (72-206; P < 0.05) and 118 (69-75; P < 0.01) fmol mg(-1) protein during prolonged oxytocin-augmented and oxytocin-induced labour, respectively. The corresponding oxytocin receptor mRNA concentrations decreased by 60- and 300-fold, respectively. The decrease in receptor binding and mRNA in women receiving oxytocin infusion indicates that homologous receptor desensitization occurs in vivo.
The role of oestradiol in the control of uterine responsiveness to oxytocin was investigated by measuring oxytocin-induced phospholipase C activation in [3H]inositol-labelled cultured human myometrial cells. Addition of oestradiol to steroid-free culture medium (10% (v/v) fetal calf serum treated with dextran-coated charcoal in phenol red-free medium) enhanced formation of inositol phosphates and this effect was completely abolished by the anti-oestrogen tamoxifen. The inhibitory effect of tamoxifen on oxytocin-induced phospholipase C activation occurred in both steroid-free and complete culture medium; it was time- and concentration-dependent and was only partly reversed by oestradiol. When phospholipase C was activated with PGF2α or fluoroaluminate instead of oxytocin, oestradiol and tamoxifen had the same stimulatory and inhibitory effects, respectively. The inhibitory effect of tamoxifen could not be prevented by treating the cells with pertussis toxin. Moreover, the effect of tamoxifen was not mediated by inhibition of protein kinase C, since the use of staurosporine (a protein kinase inhibitor) resulted in potentiation of phospholipase C activation by oxytocin. Both oestradiol and tamoxifen increased [3H]inositol incorporation into cellular lipids and cell proliferation. These results suggest that oestradiol enhances myometrial responsiveness to oxytocin and other agonists by facilitating phospholipase C activation at a post-receptor level. This effect is antagonized by tamoxifen; however, tamoxifen also has oestrogen-independent inhibitory effects.
The expression of heterotrimeric (αβγ subunits) GTP-binding regulatory proteins (G proteins) and the activation of G protein-linked receptors in human granulosa cells were investigated. The cells were obtained from stimulated follicles in women undergoing in vitro fertilization and were cultured in serum-supplemented medium. Immunoblotting with specific antibodies showed that granulosa cell membranes express αs, αi3 αi1,2, αq,11 and β subunits. Three antibodies against αo failed to detect this protein. The cells responded to hCG and to prostaglandin E2 with a dose-dependent increase in cAMP formation, confirming the functional activation of Gαs. The α2 adrenoceptor agonist, clonidine, inhibited hCG-stimulated cAMP formation and this effect was blocked with pertussis toxin, thus involving a Gi-type protein, most likely Gαi2. Oxytocin provoked an increase in formation of inositol phosphates and intracellular calcium concentration, which was partly pertussis toxin resistant, providing evidence of Gαq,11 activation. However, a significant component of the response to oxytocin could be blocked by pertussis toxin, indicating Gi-mediated phospholipase C activation (by either αi or βγ subunits). These data demonstrate the presence of G proteins in granulosa cells and suggest a complex regulation of hormonal signalling. The concentration of cAMP in these cells depended on the balance of Gαs:Gαi activation, whereas activation of the inositol phospholipid pathway and rises in intracellular calcium involved both Gq,11 and Gi pathways.