Proceedings of The Physiological Society
University of Edinburgh (2011) Proc Physiol Soc 25, C09 and PC09
Hormonal state changes the mRNA profile of calcium signaling proteins in uterine smooth muscle
G. C. Bett1,2
1. Physiology and Biophysics, SUNY, University at Buffalo, Buffalo, New York, United States. 2. Gynecology-Obstetrics, SUNY, University at Buffalo, Buffalo, New York, United States.
Calcium plays a key role in the physiology of uterine smooth muscle. Transmembrane calcium fluxes contribute to action potential shape and intracellular calcium mediates excitation contraction coupling. The electrical activity of the uterus is significantly altered throughout the estrous cycle, as well as in pregnancy. Uterine muscle is contractile, but the degree of coordinated contraction changes significantly during the estrous cycle and pregnancy. Changes in uterine contraction reflect changes in coordination of excitation due to electrical connections through gap junctions and excitatory currents, but other cellular changes may also occur during the course of pregnancy. For many ion channels, mRNA expression is altered by hormonal regulation. Therefore, the molecular basis of excitation-contraction coupling is also likely to be significantly altered depending on the state of uterine muscle. To test the hypothesis that there is substantial remodeling of cellular uterine excitation contraction coupling during different stages of estrous and during pregnancy, we undertook quantitative analysis of mRNA expression of calcium channels and other channels and related genes which affect calcium dynamics, during the estrous cycle and pregnancy. Uteri were removed from anaesthetized (inhaled isoflurane) mice (C57BL) which were regularly cycling (n=15), ovariectomized (OVX, n=10), or pregnant (not in labor, n=10). Mice were euthanized by cervical dislocation following removal of organs. mRNA was extracted (Trizol, Invitrogen and RNAeasy, Qiagen) and converted to cDNA (First strand synthesis, SABiosicences). We used quantitative real time PCR (SYBR Green in an iQ icycler, Biorad) to determine changes in relative mRNA expression. mRNA Expression was normalized to 5 housekeeping genes (beta Glucuronidase, beta, Hypoxanthine guanine phosphoribosyl transferase 1, Heat shock protein 90 alpha (cytosolic), class B member 1, Glyceraldehyde-3-phosphate dehydrogenase, and beta actin). Data were analyzed using ANOVA (p<0.05 was regarded as significant). There were significant changes in relative mRNA expression including Cacna1a (P/Q type, alpha 1A subunit, Cav2.1), Cacna1c (L type, alpha 1C subunit, Cav1.2), Cacna1g (T type, alpha 1G subunit, Cav3.1d), Cacna1h (T type, alpha 1H subunit, Cav3.2). There were also significant differences in the mRNA expression of the ancillary subunits cacnb1 (beta 1 subunit) and Cacnb4 (beta 4 subunit). Cacna1c, Cacna1g, Cacna1h all had lowest expression in pregnancy (not in labor), which is consistent with reduced excitation-contraction during this period. Conversely, inward rectifier currents, which can suppress excitation, were upregulated in pregnancy. Our observed changes in gene expression suggest that down regulation of calcium channels and up regulation of inward rectifier potassium channels help maintain uterine smooth muscle in a relaxed state in pregnancy prior to the onset of labor.
Where applicable, experiments conform with Society ethical requirements