Differential impact of acute and prolonged cAMP agonist exposure on protein kinase A activation and human myometrium contractile activity

Abstract

KEY POINTS SUMMARY Over 15 million babies are born prematurely each year with approximately 1 million of these babies dying as a direct result of preterm delivery. β 2 -adrenoreceptor agonists that act via cAMP can reduce uterine contractions to delay preterm labour, but their ability to repress uterine contractions lasts ≤ 48 hours and their use does not improve neonatal outcomes. Previous research has suggested that cAMP inhibits myometrial contractions via protein kinase A (PKA) activation, but this has yet to be demonstrated with PKA-specific agonists. We investigated the role of PKA in mediating cAMP-induced human myometrial relaxation, and the impact of prolonged cAMP elevation on myometrial contractility. Our findings suggest that PKA is not the sole mediator of cAMP-induced myometrial relaxation and that prolonged prophylactic elevation of cAMP alone is unlikely to prevent preterm labour (PTL). ABSTRACTAcute cAMP elevation inhibits myometrial contractility, but the mechanisms responsible are not fully defined and the long-term effects uncertain. These need to be defined in pregnant human myometrium before the therapeutic potential of cAMP-elevating agents in the prevention of preterm labour can be realised. In the present study, we tested the hypotheses that PKA activity is necessary for cAMP-induced myometrial relaxation and prolonged cAMP elevation can prevent myometrial contractions. Myometrial tissues obtained from term, pre-labour elective Caesarean sections were exposed to receptor-independent cAMP agonists to determine the relationship between myometrial contractility (spontaneous and oxytocin-induced), PKA activity, HSP20 phosphorylation and expression of contractionassociated and cAMP signalling proteins. Acute (1 h) application of cAMP agonists This article is protected by copyright. All rights reserved. 3promoted myometrial relaxation but this was weakly related to PKA activation. PKA-specific activator, 6-Bnz-cAMP, increased PKA activity (6.8 ± 2.0 mean fold versus vehicle; p = 0.0313) without inducing myometrial relaxation. Spontaneous myometrial contractility declined after 24 hours but was less marked when tissues were constantly exposed to cAMP agonists, especially for 8-bromo-cAMP (4.3 ± 1.2 mean fold versus vehicle; p = 0.0043); this was associated with changes to calponin, cofilin and HSP20 phosphorylated/total protein levels. Oxytocin-induced contractions were unaffected by pre-incubation with cAMP agonists despite treatments being able to enhance PKA activity and HSP20 phosphorylation. These data suggest that cAMP-induced myometrial relaxation is not solely dependent on PKA activity and the ability of cAMP agonists to repress myometrial contractility is lost with prolonged exposure. We conclude that cAMP agonist treatment alone may not prevent preterm labour.

Phenylephrine, a common cold remedy active ingredient, suppresses uterine contractions through cAMP signalling

“…However, the role of PKA in the cAMP downstream signalling pathway remains unclear in the myometrium. It has been recently reported that the exchange protein directly activated by cAMP (EPAC) rather than PKA may play the major role in cAMP-induced uterine relaxation 24 . Further studies will be required to better understand the mechanism of cAMP-induced uterine relaxations.…”

The impact of progesterone and RU-486 on classic pro-labour proteins & contractility in human myometrial tissues during 24-hour exposure to tension & interleukin-1β

“…Western blotting, immunodetection and horse radish peroxidase (HRP)-based enhanced chemiluminescence (ECL) imaging were undertaken as previously described (Lai et al , 2016); Novex® Sharp pre-stained protein standards (Life Technologies, Paisley, UK) were used as molecular weight markers. Additionally, Ponceau-stained PVDF membranes were imaged by placing them between sheets of transparency film and scanning them using a Scanjet 4850 desktop scanner (Hewlett Packard, Reading, UK) to save as Tag Image File Format (TIFF) files at 600 dots per inch (dpi) resolution.…”

“…Tissue strips were mounted to MLT0201 transducers (ADInstruments, Oxford, UK) attached to an 8channel organ bath (Panlab; Barcelona, Spain), using the polyester threads tied to them during 24 h tissue culture after glass beads had been detached. Organ bath chambers were filled with physiological saline solution (PSS; same composition as previously used (Lai et al, 2016)), which was maintained at 37 o C with 95 % O 2 /5 % CO 2 aeration. Tissues were submerged into these PSSfilled chambers, which contained combinations of 100 nM P4, 1 µM RU-486 and vehicle controls that matched their 24 h treatment conditions, immediately after securing them to their transducers.…”

Aminophylline and progesterone prevent inflammation-induced preterm parturition in the mouse†

“…After 25 minutes of oxytocin stimulation, tissues were bathed in PSS containing 60 mM KCl (KPSS) for 10 minutes to confirm viability by maximal depolarisation, after which the KPSS was replaced with PSS and tissues were bathed for a further 10 minutes before the experiment was terminated. All contractility data was analysed using LabChart 6 software (ADInstruments) to calculate mean integral tension values as previously described [22]. Spontaneous contractile output was measured from the 30-minute period immediately before oxytocin/H 2 O addition to tissues.…”