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Human Myometrial Quiescence and Activation during Gestation and Parturition Involve Dramatic Changes in Expression and Activity of Particulate Type II (RII) Protein Kinase A Holoenzyme

Department of Obstetrics and Gynaecology, School of Surgical and Reproductive Sciences, University of Newcastle upon Tyne, Royal Victoria Infirmary, Newcastle upon Tyne NE1 4LP, United Kingdom

Address all correspondence and requests for reprints to: G. Nicholas Europe-Finner, M.D., Department of Obstetrics and Gynaecology, School of Surgical and Reproductive Sciences, University of Newcastle upon Tyne, 4th Floor Leazes Wing, Royal Victoria Infirmary, Newcastle upon Tyne NE1 4LP, United Kingdom. E-mail: g.n.europe-finner{at}ncl.ac.uk.

There are substantial data indicating that components of the cAMP-signaling pathway are differentially expressed in the human myometrium during pregnancy. The effects of cAMP in most tissues and cell types are mainly modulated via protein kinase A, a heterotetrameric protein complex consisting of two regulatory (R) and two catalytic (C) subunits. In the studies presented here, we used specific antibodies in Western blotting/immunoprecipitation, RT-PCR, and functional protein kinase A (PKA) phosphorylation assays to determine the PKA holoenzymes that are expressed in the human myometrium throughout pregnancy and labor. We report that as early as the second trimester of pregnancy, there is a significant increase in expression of the regulatory RII protein subunit of PKA in the myometrium. This increase in protein expression is also mirrored at the mRNA level, indicating transcriptional control throughout pregnancy, whereas during parturition both transcript and protein are significantly decreased. This increase in RII protein also resulted in increased particulate PKA activity in the myometrium during gestation, which was subsequently decreased during labor. Two specific A kinase anchoring proteins, AKAP95 and AKAP79, which have high binding affinities for RII subunits, were found to form complexes with myometrial RII species employing immunoprecipitation assays, but their levels of expression remained uniform in all myometrial tissue samples investigated. Our findings indicate that increased particulate type II PKA activity occurs throughout pregnancy, therefore directing the cAMP quiescence signal to specific subcellular loci within myometrial smooth muscle cells including the contractile machinery at the cytoskeleton; this effect is then removed during parturition.

This work was supported by a grant from Action Research (SP3689).

M.W.J.M. and G.N.E.-F. contributed equally to this study.

Abbreviations: AKAP, A kinase-anchoring protein; C, catalytic subunit; CPT, 8, 4-chlorophenylthio; ECL, enhanced chemiluminescence; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NP, nonpregnant; P, term pregnant; PKA, protein kinase A; R, regulatory subunit; SL, term spontaneously laboring; TBS, Tris-buffered saline.

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