Functional and immunohistochemical evidence for the presence of extracellular calcium-sensing receptors in the oestrogen-dominated rat’s uterus

Marc Pistilli1, James J Petrik2, Alison C Holloway1, Denis J Crankshaw1

1McMaster University, Hamilton, ON, Canada, 2University of Guelph, Guelph, ON, Canada

The extracellular calcium-sensing receptor (CaR) is a G protein-coupled receptor that was originally identified in bovine parathyroid gland (Brown et al, 1993). Subsequently, CaRs have been shown to be expressed in the endothelium and adventitia of blood vessels where they mediate relaxation by both endothelium-dependent and endothelium-independent mechanisms (Weston et al, 2005; Geraghty et al, 2007; Smajilovic et al, 2007). CaRs may serve a more general role as regulators of smooth muscle contractility, but the function and expression of CaRs has yet to be documented in uterus.

We hypothesised that CaRs are expressed in uterine smooth muscle where they mediate inhibition of contractility. The two main objectives of this study were to determine the effects of several CaR agonists and one positive allosteric modulator on the contractility of the rat’s uterus in vitro, and to confirm the presence of CaRs in the tissue by immunohistochemistry.

Female Wistar rats were primed with diethylstilboestrol to maintain a constant hormonal state and longitudinal uterine strips were isolated and set up in organ baths as described by Crankshaw (2001), except that the extracellular calcium concentration was 0.5 mM. Contractions were driven with oxytocin (1 nM), allowing for a 10 minute control recording, and the following CaR ligands, dissolved in deionized water, were added cumulatively in half log concentration increments to individual uterine strips, allowing for a 2 minute equilibrium time followed by a 10 minute recording: calcium, cinacalcet (DMSO solvent), gadolinium, neomycin, spermidine and spermine. Responses were expressed as a percent of the oxytocin driven contractions. Inhibitory potencies were expressed as pIC50 values which were obtained from concentration-inhibition curves. Uteri from similar animals were fixed, prepared, and reacted with anti-CaR antibody to identify the presence of CaR immunoreactivity.

All the CaR ligands tested inhibited oxytocin-driven uterine contractions fully. Spermine and spermidine had biphasic effects, causing contraction at higher concentrations. The inhibitory potency order was: cinacalcet > gadolinium > spermine > spermidine > neomycin > calcium. The selective CaR positive allosteric modulator cinacalcet had a pIC50 value of 5.6 ± 0.4 (n = 3), which is in good agreement with its potency in aortic smooth muscle (Smajilovic et al, 2007). CaR immunostaining was detected in both the longitudinal and circular layers of the myometrium.

The effects of the CaR ligands provide strong evidence for functional CaRs coupled to inhibition of contractility in rat uterus. The confirmation of antibody staining on the myometrial layers confirms the physical presence of the receptor, while the control conditions in the absence of primary antibody negate the possibility of non-specific binding.

Supported by SickKids Foundation & Canadian Institutes of Health Research

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