Use of antisense oligonucleotides to verify the role of the alpha(1A)-adrenergic receptor in the contractility of the rat uterus post partum.

Abstract

The adrenergic system plays a major role in the regulation of the contractility of the uterus during pregnancy. This study investigated the role of the alpha(1A)-adrenergic receptor (AR) in this regulation. The use of partial phosphorothioate antisense oligodeoxynucleotides (AONs) permitted the sequence-selective inhibition of AR gene expression. AONs were injected together with a cationic liposomal carrier agent into the post partum rat uterus. Incubation for 12 or 24 h with the most effective AON (480-AON) caused a 58.7 or 53.0% inhibition, respectively, of the expression of the alpha(1A)-AR density, whereas incubation for 36 or 48 h resulted in only a 38.8 or 26.7% inhibition, respectively. The decrease of the alpha(1A)-AR density by 480-AON was demonstrated by Western blot analysis and a radioreceptor binding assay on rat uterus preparations 24 h after delivery. The changes in the contractility of the uterus after AON treatment were measured on isolated rat uterine tissue by electric field stimulation. The significant decrease in the ability of the uterus to contract was indicated by the area under the curve method. The electric field studies revealed that the specific alpha(1A)-blockers 5-methylurapidil and WB 4101 inhibited the rhythmic contraction by about 74 and 70% in the control uteri and by 25 and 20% in 480-AON-treated uteri, respectively. The curves for the beta-mimetic (terbutaline) and alpha(1D)-antagonist (BMY7370) inhibitors were unchanged after 480-AON treatment of the uteri. These results suggest the importance of the alpha(1A)-AR in the tocolytic effect exerted by the alpha(1)-antagonist, although high concentrations of antagonists can not exclude the role of alpha(1D)-ARs, too. Additionally, these prove that the knockdown transformation by AONs offers a useful animal model for the investigation of receptors controlling the function of uterine tissue.