Eng-Cheng Chan

Postnatal disappearance of the pregnancy-associated reduced sensitivity of plasma cortisol to feedback inhibition.

We recently observed that the characteristic insensitivity of the pituitary-adrenal system in women to feedback inhibition during pregnancy persists for at least four days postnatally. We therefore examined women during the first five weeks after delivery to assess when the sensitivity of plasma cortisol to glucocorticoid inhibition returns to normal. Dexamethasone (DEXA, 1 mg) was ingested at 11 pm by normal healthy women, once between the 3rd and 27th postnatal days, and again on day 35. Blood plasma was collected at 4 pm on the following day for cortisol assay. Plasma cortisol levels (nmol/L, mean +/- sem [n]) after DEXA in the first two weeks (216 +/- 28, [47]) were higher (p less than 0.001) than in nonmedicated nonpregnant women (47.4 +/- 8.9 [12]) and were normal by the 35th day after delivery (41.7 +/- 4.8 [74]). A negative association was found between post-DEXA cortisol and time after delivery in the first 4 post-partum weeks (r = -0.46, p less than 0.001). The study confirms that insensitivity of plasma cortisol to feedback inhibition persists beyond normal pregnancy in a significant proportion of healthy women for two to three weeks, and is absent by the 5th postnatal week.

G-1-Activated Membrane Estrogen Receptors Mediate Increased Contractility of the Human Myometrium

Estrogens are key mediators of increased uterine contractility at labor. We sought to determine whether membrane-associated estrogen receptors, such as the recently described seven-transmembrane receptor G protein-coupled receptor 30 (GPR30), mediated some of this effect. Using human myometrium obtained at term cesarean section before or after the onset of labor, we demonstrated the presence of GPR30 mRNA and protein using quantitative RT-PCR and Western blotting. GPR30 receptor was localized to the cell membrane and often colocalized with calveolin-1. Using the specific estrogen membrane receptor agonist G-1 and myometrial explants, we showed that membrane receptor activation led to phosphorylation of MAPK and the actin-modifying small heat shock protein 27. Using myometrial strips incubated with G-1 or vehicle we demonstrated that estrogen membrane receptor activation increased the myometrial contractile response to oxytocin. These data suggest that activation of the plasma membrane estrogen receptor GPR30 likely participates in the physiology of the human myometrium during pregnancy and identifies it as a potential target to modify uterine activity.

Role of serine-threonine phosphoprotein phosphatases in smooth muscle contractility

The degree of phosphorylation of myosin light chain 20 (MLC20) is a major determinant of force generation in smooth muscle. Myosin phosphatases (MPs) contain protein phosphatase (PP) 1 as catalytic subunits and are the major enzymes that dephosphorylate MLC20. MP regulatory targeting subunit 1 (MYPT1), the main regulatory subunit of MP in all smooth muscles, is a key convergence point of contractile and relaxatory pathways. Combinations of regulatory mechanisms, including isoform splicing, multiple phosphorylation sites, and scaffolding proteins, modulate MYPT1 activity with tissue and agonist specificities to affect contraction and relaxation. Other members of the PP1 family that do not target myosin, as well as PP2A and PP2B, dephosphorylate a range of proteins that affect smooth muscle contraction. This review discusses the role of phosphatases in smooth muscle contractility with a focus on MYPT1 in uterine smooth muscle. Myometrium shares characteristics of vascular and other visceral smooth muscles yet, during healthy pregnancy, undergoes hypertrophy, hyperplasia, quiescence, and labor as physiological processes. Myometrium presents an accessible model for the study of normal and pathological smooth muscle function, and a better understanding of myometrial physiology may allow the development of novel therapeutics for the many disorders of myometrial physiology from preterm labor to dysmenorrhea.

Differential processing of corticotrophin-releasing hormone by the human placenta and hypothalamus.

The molecular forms of corticotrophin-releasing hormone (CRH) in human placentae and hypothalami were investigated by gel permeation chromatography of water extracts. Hypothalamic extracts produced one peak of immunoreactivity which coeluted with human CRH at Kd = 0.53. Placental extracts, however, had in addition to that peak, two other peaks eluting earlier at the void and at Kd = 0.35-0.38. Tryptic digestion of the middle peak produced immunoreactivity which coeluted with the standard. Larger forms were also found in plasma of women in the third trimester of pregnancy and during labour but not in eluates from superfused placental fragments which had only CRH41-sized material. These data indicate that tissue-specific post-translational processing occurs for CRH, and suggests that the link between synthesis and secretion is more immediate in the placenta than hypothalamus.

Evidence that a protein kinase A substrate, small heat-shock protein 20, modulates myometrial relaxation in human pregnancy

For a successful human pregnancy, the phasic smooth muscle of the myometrium must remain quiescent until labor. Activation of cAMP/cAMP-dependent protein kinase A (PKA) pathways contributes to this quiescence. The small heat-shock protein 20 (HSP20) is a target of PKA, and phosphorylated HSP20 (pHSP20) modulates relaxation of tonic vascular smooth muscle via interaction with actin, independent of myosin dephosphorylation. Our objective was to determine whether relaxation in human myometrium is associated with changes in phosphorylation of HSP20. Myometrium was obtained at elective cesarean. Elevating cAMP with forskolin or rolipram (a phosphodiesterase inhibitor) caused substantial relaxation of spontaneously contracting human myometrial strips, of 92 +/- 4% (mean +/- sem, n = 10) and 84 +/- 7% (n = 6), respectively. Subsequent two-dimensional electrophoresis with immunoblotting of strip extracts showed a significant 2.6- and 2.1-fold increase in phosphorylated HSP20 (pHSP20) after forskolin (P < 0.01; n = 5) or rolipram treatment (P < 0.05; n = 4). Noncyclic-nucleotide-mediated relaxation, induced by the calcium channel blocker nifedipine, did not alter pHSP20. Inhibition of PKA with H89 significantly attenuated rolipram-induced relaxation (P < 0.01; n = 4), and partially reduced rolipram-stimulated pHSP20. Total and pHSP20 protein was unchanged in term laboring and nonlaboring myometria. Coimmunoprecipitation studies revealed a specific association of HSP20 with alpha-smooth muscle actin and HSP27, a key regulator of actin filament dynamics. Finally, coimmunofluorescence demonstrated moderate colocalization of HSP20 with alpha-smooth muscle actin in the cytoplasm of laboring myometria. Our data support a novel role for pHSP20 in the modulation of cyclic-nucleotide-mediated myometrial relaxation, through interaction with actin. pHSP20 represents an important new target for future tocolytic therapy.

Secretion of corticotropin-releasing hormone by superfused human placental fragments

Corticotropin-releasing hormone (CRH) immunoreactivity (IR) is present in the blood of women in the 3rd trimester of pregnancy and in placental extracts. We have used a placental fragment superfusion system to investigate the release of CRH from fresh placental tissue. Fragments of normal term placenta were mixed with Biogel P2, packed into minicolumns and superfused with carbogen-gassed Earles buffer at 37 degrees C. The rheology of the superfusion system was determined and the oxygen consumption of the superfused placental fragments indicated viability of the tissue preparation over a 5-hour time span. CRH IR in the eluate was measured by radioimmunoassay (RIA) using the 41 residue synthetic peptide human, rat CRH-41 (h, r CRH-41) as the standard, 125I labelled Tyr- h, r CRH as the tracer and rabbit anti-ovine CRH as the antibody. The sensitivity of the assay is 2 pM. Size exclusion chromatography on Sephadex G-50 of the placental column eluate displayed one major peak of CRH IR which co-eluted with that of h, r CRH. Placental fragment superfusate displayed potent CRH bioactivity as assessed by beta-endorphin secretion from ovine pituitary cells. Replacing the superfusing medium of the placental fragments with 45 mM KCl resulted in a prompt increase in the release of CRH IR. These results indicate that placental cells in vitro secrete a molecule of similar molecular weight, immunoreactivity and bioactivity to h, r CRH and that the rate of secretion may be regulated.

Diminished hERG K+ channel activity facilitates strong human labour contractions but is dysregulated in obese women

Human ether-a-go-go-related gene (hERG) potassium channels determine cardiac action potential and contraction duration. Human uterine contractions are underpinned by an action potential that also possesses an initial spike followed by prolonged depolarization. Here we show that hERG channel proteins (α-conducting and β-inhibitory subunits) and hERG currents exist in isolated patch-clamped human myometrial cells. We show that hERG channel activity suppresses contraction amplitude and duration before labour, thereby facilitating quiescence. During established labour, expression of β-inhibitory protein is markedly enhanced, resulting in reduced hERG activity that is associated with an increased duration of uterine action potentials and contractions. Thus, changes in hERG channel activity contribute to electrophysiological mechanisms that produce contractions during labour. We also demonstrate that this system fails in women with elevated BMI, who have enhanced hERG activity as a result of low β-inhibitory protein expression, which likely contributes to the weak contractions and poor labour outcomes observed in many obese women necessitating caesarean delivery.

Phasic Phosphorylation of Caldesmon and ERK 1/2 during Contractions in Human Myometrium

Human myometrium develops phasic contractions during labor. Phosphorylation of caldesmon (h-CaD) and extracellular signal-regulated kinase 1/2 (ERK 1/2) has been implicated in development of these contractions, however the phospho-regulation of these proteins is yet to be examined during periods of both contraction and relaxation. We hypothesized that protein phosphorylation events are implicated in the phasic nature of myometrial contractions, and aimed to examine h-CaD and ERK 1/2 phosphorylation in myometrium snap frozen at specific stages, including; (1) prior to onset of contractions, (2) at peak contraction and (3) during relaxation. We aimed to compare h-CaD and ERK 1/2 phosphorylation in vitro against results from in vivo studies that compared not-in-labor (NIL) and laboring (L) myometrium. Comparison of NIL (n = 8) and L (n = 8) myometrium revealed a 2-fold increase in h-CaD phosphorylation (ser-789; P = 0.012) during onset of labor in vivo, and was associated with significantly up-regulated ERK2 expression (P = 0.022), however no change in ERK2 phosphorylation was observed (P = 0.475). During in vitro studies (n = 5), transition from non-contracting tissue to tissue at peak contraction was associated with increased phosphorylation of both h-CaD and ERK 1/2. Furthermore, tissue preserved at relaxation phase exhibited diminished levels of h-CaD and ERK 1/2 phosphorylation compared to tissue preserved at peak contraction, thereby producing a phasic phosphorylation profile for h-CaD and ERK 1/2. h-CaD and ERK 1/2 are phosphorylated during myometrial contractions, however their phospho-regulation is dynamic, in that h-CaD and ERK 1/2 are phosphorylated and dephosphorylated in phase with contraction and relaxation respectively. Comparisons of NIL and L tissue are at risk of failing to detect these changes, as L samples are not necessarily preserved in the midst of an active contraction.

Pathological interactions with the timing of birth and uterine activation

The physiological processes that regulate the onset of parturition and birth are slowly being elucidated, and the points at which pathology can intervene are becoming more apparent. The data support the view that multiple pathways lead to myometrial activation. The clinical corollary is that combinations of tocolytics that operate via different mechanisms may be more effective than single agents. It may also be necessary to divide preterm labour into groups based on underlying mechanisms and to tailor therapy accordingly.

Desensitization of Superfused Isolated Ovine Anterior Pituitary Cells to Human Corticotropin‐Releasing Factor

We have employed an in vitro system to study the time‐course of β‐endorphin (β‐EP) immunoreactivity release from anterior pituitary cells stimulated with corticotropin‐releasing factor (CRF) and whether exposure to CRF desensitizes the cells to subsequent stimulation. Ovine anterior pituitaries were enzymatically disrupted into single cells, mixed with Siegel P2 and superfused in mini‐columns with carbogen‐gassed medium at 37 °C. Superfusate fractions were collected at 5‐min intervals and β‐EP immunoreactivity in the eluate was measured by radioimmunoassay. Peaks of β‐EP release that rose significantly above baseline noise were detected using the PULSAR algorithm.