Proceedings of The Physiological Society

University of Oxford (2005) J Physiol 568P, PC18

Poster Communications

Molecular expression of ion channel subtypes in human endometrium

Patel, Suraj; Shaw, Robert W; Arya, Pratibha; Warren, Averil; Khan, Raheela N;

1. Academic Division of Obstetrics & Gynaecology, University of Nottingham, Derby, United Kingdom. 2. Derby City General Hospital, Derby, United Kingdom.

The adult human endometrium, a unique tissue undergoing monthly cellular remodelling in preparation for implantation, consists predominantly of epithelial and stromal cells. Ion channels of non-excitable cells are involved in diverse cellular functions that include proliferation and secretion. Endometrial function is thus likely to involve stage specific expression of ion channel. The four transmembrane domain two pore potassium (K2P) channels are a family of K+ channels that contribute to the resting membrane potential and may be regulated by various factors including heat, stretch and pH. A second class of channels of interest is the transient receptor potential vanilloid (TRPV) subfamily, in particular TRPV5 and TRPV6 which mediate Ca2+ entry in epithelial cells. The aim of this investigation was to determine ion channel expression of known subtypes in human endometrium. This study had local ethics committee approval. Endometrial samples were obtained by pipelle biopsy from healthy, non-pregnant, fertile women (<45 years of age), during clinical investigation for benign disorders. All women gave fully informed written consent. Total RNA was extracted from snap-frozen endometria using TRIzol reagent and reverse-transcribed to cDNA. Reverse transcriptase-polymerase chain reaction (RT PCR) was carried out using primers specific to members of the K2P channel family, TRPV5, TRPV6 and the amiloride sensitive Na+ channel. For immunofluorescence, endometrial samples were separated into stromal and glandular cells by selective filtration and cultured overnight. Thereafter cells were fixed and processed for cytokeratin, vimentin and TWIK-2 immunostaining using the avidin-biotin complex (ABC) technique. Of the K2P channels tested, PCR products of the expected size were observed for TWIK-2 (n = 16/16), TREK-1 (n = 3/6), KCNK7 (n = 19/23). There was no expression for TRAAK or TASK-1. TRPV6, but not TRPV5, was expressed in all samples (n = 6/6) as was the case for the amiloride-sensitive Na+ channel (n = 6/6). All samples tested positive for the endometrial epithelial cell marker E cadherin and the housekeeping gene β-actin. Cytokeratin and vimentin immunoreactivity was observed in epithelial and stromal cells respectively. TWIK-2 specific immunofluorescence was apparent in both cell types. Our findings demonstrate the presence of mRNA for TREK-1, KCNK7, TWIK-2, and TRPV6 in the human endometrium. Immunofluorescence revealed translation of TWIK-2 mRNA into protein in both stromal and glandular endometrial cells. Expression of TRPV6 is in agreement with findings from other epithelial cells possibly indicating their importance in regulating Ca2+ entry in endometrium (1). Studies are continuing to determine if expression is temporally and functionally associated with samples obtained during the proliferative or secretory phase of the menstrual cycle.

Where applicable, experiments conform with Society ethical requirements