cAMP Compartmentalisation in Human Myometrial Cells.

Preterm birth is the leading cause of childhood mortality and morbidity. A better understanding of the processes that drive the onset of human labour is essential to reduce the adverse perinatal outcomes associated with dysfunctional labour. Beta-mimetics, which activate the myometrial cyclic adenosine monophosphate (cAMP) system, successfully delay preterm labour, suggesting a key role for cAMP in the control of myometrial contractility; however, the mechanisms underpinning this regulation are incompletely understood. Here we used genetically encoded cAMP reporters to investigate cAMP signalling in human myometrial smooth muscle cells at the subcellular level. We found significant differences in the dynamics of the cAMP response in the cytosol and at the plasmalemma upon stimulation with catecholamines or prostaglandins, indicating compartment-specific handling of cAMP signals. Our analysis uncovered significant disparities in the amplitude, kinetics, and regulation of cAMP signals in primary myometrial cells obtained from pregnant donors compared with a myometrial cell line and found marked response variability between donors. We also found that in vitro passaging of primary myometrial cells had a profound impact on cAMP signalling. Our findings highlight the importance of cell model choice and culture conditions when studying cAMP signalling in myometrial cells and we provide new insights into the spatial and temporal dynamics of cAMP in the human myometrium.

Mechanical heart valves and pregnancy: Issues surrounding anticoagulation. Experience from two obstetric cardiac centres.

BACKGROUND: Pregnant women with mechanical heart valves are at significant risk of obstetric/cardiac complications. This study compares the anticoagulation management in two obstetric cardiac centres. METHODS: Retrospective case-note review from Chelsea and Westminster/Royal Brompton Hospitals (CR) and Erasmus Medical Centre (EMC). Main outcome measure was mechanical heart valve thrombosis. RESULTS: Nineteen pregnancies from CR and 25 pregnancies from EMC were included. Most women were on low-molecular-weight heparin (LMWH) throughout pregnancy at CR, whereas at EMC most had LMWH in the first trimester and vitamin K antagonists in subsequent trimesters. Peak anti-factor Xa were performed monthly at CR, levels 0.39-1.51 IU/mL (mean 0.82 IU/mL). Anticoagulation management peri-partum was inconsistent. Delivery was mainly by caesarean section at CR (74%) and vaginal delivery at EMC (64%). No maternal deaths and only one mechanical heart valve thrombosis at CR. Two mechanical heart valve thromboses and one maternal death at EMC. CONCLUSION: Peri-partum anticoagulation strategies, anticoagulation monitoring and mode of delivery inconsistencies reported.

PKA and AKIP1 interact to mediate cAMP-driven COX-2 expression: A potentially pivotal interaction in preterm and term labour.

Previously, we showed that cAMP increased COX-2 expression in myometrial cells via MAPK. Here, we have extended these observations, using primary myometrial cell cultures to show that the cAMP agonist, forskolin, enhances IL-1ß-driven COX-2 expression. We then explored the role of A-kinase interacting protein (AKIP1), which modulates the effect of PKA on p65 activation. AKIP1 knockdown reversed the effect of forskolin, such that its addition inhibited IL-1ß-induced COX-2 mRNA expression and reduced the IL-1ß-induced increase in nuclear levels of p65 and c-jun. Forskolin alone and with IL-1ß increased IκBα mRNA expression suggesting that in the context of inflammation and in the presence of AKIP1, cAMP enhances p65 activation. AKIP1 knockdown reversed these changes. Interestingly, AKIP1 knockdown had minimal effect on the ability of forskolin to repress either basal OTR expression or IL-1ß-stimulated OTR mRNA expression. AKIP1 was up-regulated by IL-1ß, but not stretch and was repressed by cAMP. The mRNA expression of AKIP1 increased in early labour in tandem with an increase in COX-2 mRNA and protein. AKIP1 protein levels were also increased with inflammation and stretch-induced preterm labour. Our results identify a second important cAMP effector-switch occurring at term in human myometrium and suggest that a hitherto unrecognized interaction may exist between AKIP1, NFκB and AP-1. These data add to the proposition that cAMP acts as a key regulator of human myometrial contractility.

The interaction between protein kinase A and progesterone on basal and inflammation-induced myometrial oxytocin receptor expression.

Our previous work has shown myometrial PKA activity declines in term and twin-preterm labour in association with an increase in the expression of the oxytocin receptor (OTR). Here we investigate the action of cAMP/PKA in basal conditions, with the addition of progesterone (P4) and/or IL-1ß to understand how cAMP/PKA acts to maintain pregnancy and whether the combination of cAMP and P4 would be a viable therapeutic combination for the prevention of preterm labour (PTL). Further, given that we have previously found that cAMP enhances P4 action we wanted to test the hypothesis that changes in the cAMP effector system are responsible for the functional withdrawal of myometrial P4 action. Myometrial cells were grown from biopsies obtained from women at the time of elective Caesarean section before the onset of labour. The addition of forskolin, an adenylyl cyclase activator, repressed basal OTR mRNA levels at all doses and P4 only enhanced this effect at its highest dose. Forskolin repressed the IL-1ß-induced increase in OTR mRNA and protein levels in a PKA-dependent fashion and repressed IL-1ß-activation and nuclear transfer of NFκB and AP-1. P4 had similar effects and the combination P4 and forskolin had greater effects on OTR and NFκB than forskolin alone. While PKA knockdown had no effect on the ability of P4 to repress IL-1ß-induced OTR expression it reversed the repressive effect of the combination of P4 and forskolin and resulted in a greater increase than observed with IL-1ß alone. These studies suggest that cAMP acts via PKA to repress inflammation-driven OTR expression, but that when PKA activity is reduced, the combination of cAMP and P4 actually enhances the OTR response to inflammation, promoting the onset of labour and suggesting that changes in the cAMP effector system can induce a functional P4 withdrawal.

Changes in cAMP effector predominance are associated with increased oxytocin receptor expression in twin but not infection-associated or idiopathic preterm labour.

We previously reported that at term pregnancy, a decline in myometrial protein kinase A (PKA) activity leads to an exchange protein activated by cyclic AMP (Epac1)-dependent increase in oxytocin receptor (OTR) expression, promoting the onset of labour. Here, we studied the changes in the cyclic adenosine monophosphate (cAMP) effector system present in different phenotypes of preterm labour (PTL). Myometrial biopsies obtained from women with phenotypically distinct forms of PTL and the levels of PKA and OTR were examined. Although we found similar changes in the cAMP effector pathway in all forms of PTL, only in the case of twin PTL (T-PTL) was myometrial OTR levels increased in association with these results. Although there were several changes in the mRNA levels of components of the cAMP synthetic pathway, the total myometrial cAMP levels did not change with the onset of any subtype of PTL. With regards to the expression of cAMP-responsive genes, we found that the mRNA levels of 4 of the 5 cAMP-down-regulated genes were increased in T-PTL, similar to our findings in term labour. These data signify that although changes in the cAMP effector system were common to all forms of PTL, only in T-PTL were OTR levels increased. Similarly, the mRNA levels of cAMP-repressed genes were only increased in T-PTL supporting the concept that the decline in PKA levels influences myometrial function driving the onset of T-PTL.

Evidence-based first aid advice for paediatric burns in the United Kingdom.

BACKGROUND: Burn and scald injuries are common in children. First aid advice for paediatric burns is offered by a range of health organisations and charities in the UK. Despite this, children still present to emergency departments and burn services having received little or inadequate first aid. METHOD: A survey was undertaken regarding the content and consistency of the advice given by a cross-section of UK health organisations involved in first aid prevention and education. The advice was subsequently examined to determine if it was evidence-based. RESULTS: Our study has demonstrated inconsistencies in the content of the first aid advice provided by the 21 organisations included in the study. Seventy-one percent of the information was only available online. The temperature, method and duration of cooling varied substantially, as did the advice recommended for the removal of clothing and jewellery and methods for covering the burn immediately after injury. Results from the literature review concluded the following based on available evidence; cool the burn with running tap water for 20min, remove clothing and jewellery and cover the burn with cling film or a clean non-adhesive dressing. CONCLUSIONS: This study highlights the lack of consistency between first aid guidance provided by health organisations and charities in the UK.