We describe here the expression of the ryanodine receptor isoforms RyR2 and RyR3 in human non-pregnant and pregnant (non-labouring) myometrium, and in isolated cultured myometrial cells. The mRNA encoding the RyR3 isoform was found in both non-pregnant and pregnant myometrial tissue samples; however, the mRNA for RyR2 was found only in pregnant samples. It can be speculated that the appearance of this additional isoform in the pregnant myometrium may increase the ability of this tissue to contract at term. Control of expression of the RyR2 gene may therefore be another example of an up-regulated signalling system in pregnancy. Although the mRNA for RyR3 was expressed in cultured myometrial cells, the mRNA for RyR2 could not be detected. Thus cultured myometrial cells appear to be similar to the non-pregnant myometrium. The cytokine transforming growth factor beta (TGF-beta) has been reported to alter RyR mRNA expression in many cell types. After treatment with TGF-beta, both RyR2 and RyR3 mRNAs could be detected in cultured myometrial cells. These observations support the idea that the expression of the RyR2 isoform is up-regulated both in pregnancy and in TGF-beta-treated cultured myometrial cells. Using measurements of 45Ca2+ release, we have further demonstrated that cultured human myometrial cells show a significant augmentation of both the Ca2+-induced Ca2+ release (CICR) mechanism and ryanodine-induced Ca2+ release after treatment with TGF-beta. Additionally, caffeine was able to induce Ca2+ release and sensitize the CICR mechanism to ryanodine. Thus we suggest that the appearance of RyR2 mRNA leads to the expression of this receptor/channel protein with identifiable pharmacological characteristics. These results are discussed in the context of the potential role of gene activation in the process of maturation of the human myometrium during pregnancy.
Partial cDNAs of the ryanodine receptor were cloned using PCR analysis from reverse transcribed total and mRNA, extracted from freshly isolated pregnant, non-pregnant, and cultured human myometrial smooth muscle. The identity of these clones was confirmed by nucleotide sequencing of the fragments and indicate the expression of both the skeletal and brain ryanodine receptor isoforms in these preparations. In freshly isolated non-pregnant myometrial tissue, membrane fractions displaying specific [3H]ryanodine binding activities were isolated using density gradient centrifugation. SDS-PAGE of the sucrose gradient fractions indicated the specific comigration of a polypeptide with a molecular mass of~544 kDa with the ryanodine binding activity.
In cultured human myometrial cells application of caffeine (1–30 mM) did not result in an elevation of intracellular Ca2+ ([Ca2+]i). Caffeine was found to reversibly inhibit both spontaneous and agonist‐induced repetitive rises in [Ca2+]i possibly as a consequence of its ability to interfere with the binding of inositol trisphosphate (IP3) to the receptor on the sarcoplasmic reticulum. Brief applications of ryanodine (1–10μM) were observed to elevate [Ca2+]i and repeated exposures to ryanodine could elicit Ca2+ transients of similar magnitude. Ryanodine was also observed to mobilise Ca2+ in cells bathed in nominally Ca2+‐free solution. These observations suggest the presence of a novel type of ryanodine‐sensitive Ca2+‐induced Ca2+ release (R‐CICR) system in human myometrial cells.
The mechanisms responsible for the mobilisation of Ca2+ from intracellular stores sensitive to inositol trisphosphate (InsP3) were studied in saponin-permeabilised human myometrial cells in which the sarcoplasmic reticulum was pre-loaded with 45Ca2+. InsP3-induced 45Ca2+ release was measured over the InsP3 concentration range of 100 nM to 100 microM and showed a graded response. InsP3-induced 45Ca2+ release was inhibited by heparin (20-40 microg/ml) but not significantly affected by caffeine. The Ca2+ sensitivity of InsP3-induced Ca2+ release was measured under conditions which were designed to exclude interference with Ca2+ released by the ryanodine receptor/channel complex. The data showed a bell-shaped relationship with the InsP3 receptor (InsP3R) functional at 10 nM, becoming maximally activated at 300 nM but inhibited at 10 microM Ca2+. Messenger RNA encoding for three isoforms of InsP3R, type I, II and type III, was shown to be present. The relative expression levels of these messengers were obtained by ratio-PCR analysis and the levels of expression of the different isoforms were found to differ between individual patients.
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