TRPV4 Is Expressed In The Uterus Of Non-pregnant Mice, But Does Not Regulate Oxytocin Responses

A. Shah, R. Patel, R.M. Tribe, A.D. Grant. King\'s College London, London, UK

The mechanosensitive Ca2+ permeable cation channel Transient Receptor Potential Vanilloid 4 (TRPV4) regulates smooth muscle contraction in the vasculature (1) and bladder (2) in response to tissue stretch. Myometrial contractions are also regulated by stretch (3), but to our knowledge the expression and function of TRPV4 in regulating murine uterine activity has not yet been investigated. We hypothesised that TRPV4 is expressed in the uterus, and regulates contractions in response to oxytocin.

Non-pregnant, virgin wild type (+/+) and TRPV4 knockout (-/-) female mice in oestrous were used in this study. Mice were sacrificed with sodium pentobarbital and uteri collected. Expression of TRPV4 was assessed by immunohistochemistry on fresh-frozen sections and by Western blotting using uterine lysates. For functional in vitro studies, uterine horns were dissected free and the endometrium was removed. Myometrial strips were suspended in Krebs-Henseleit (KH) filled organ baths and allowed to equilibrate under a resting tension of 1.0 g for 45 min. Changes in isometric force were recorded using a Power Lab v.4 system (AD Instruments, UK). Following equilibration, oxytocin concentration-response data were obtained by cumulatively adding oxytocin (1 pM to 0.3 µM) at 10 min intervals. In control experiments, KH solution was added cumulatively to the organ baths. Data were analysed for maximal amplitudes during the concentration-response period and expressed as a percentage change of spontaneous maximal contractions compared to the period preceding the oxytocin challenge. Spontaneous activity prior to treatment was normalised to the mass of the respective tissue strips, and values represented as contraction (g) per mg of tissue mass. Data are stated as mean ± S.E.M.

TRPV4 was detected in uterine lysate from TRPV4 +/+ mice, but not in lysate from TRPV4 -/- animals by western blotting. TRPV4 immunoreactivity was observed in the endometrium, myometrium and the endometrial glands of TRPV4 +/+ but not -/- mice. In vitro experiments revealed that myometrial strips from TRPV4 +/+ and -/- mice exhibited spontaneous contractions; there was no effect of TRPV4 deletion on this activity (Maximum amplitudes +/+: 0.06±0.006 vs. -/-: 0.06±0.01 g/mg). Cumulative oxytocin additions had little, if any, effect on spontaneous activity in either genotype compared to vehicle. Maximal contractions to oxytocin (0.3 µM) were unchanged in either genotype compared to control (+/+: treated vs. control 12±4 vs. 12±10%, respectively & -/-: treated vs. control 11±12% vs. -10±0%, respectively).

We conclude that TRPV4 is expressed in the murine uterus, but does not regulate basal, spontaneous uterine contraction in tissue from non-pregnant, virgin mice. The effects of oxytocin on contraction were minor, and unaffected by deletion of TRPV4. The functions of uterine TRPV4 remain to be determined, and may be worth investigating in the pregnant uterus.

1. Filosa, J.A. et al. (2013) TRPV4 and the regulation of vascular tone. J. Cardiovasc. Pharmacol. 61(2): 113-9.

2. Gevaert, T. et al. (2007) Deletion of the transient receptor potential cation channel TRPV4 impairs murine bladder voiding. J. Clin. Invest. 117(11): 3453-62.

3. Arthur, P. et al. (2007) Oxytocin and parturition: a role for increased myometrial calcium and calcium sensitization? Front. Biosci. 12: 619-33.