Preparation of cultured smooth muscle cells from human myometrium for X-ray microanalysis.

Methodological aspects of the use of X-ray microanalysis in physiological and pharmacological experiments on cultured myometrial cells were investigated. Cultured human myometrial cells were grown from biopsies after detaching the fibroblasts. Of the cultured cells, 95-98% showed desmin-like immunoreactivity. Transmission electron microscopy showed that subcultured cells were different from myometrial cells in situ. The effects of washing the cells to remove external salt-rich medium were investigated. All solutions removed the external medium, resulting in lower concentrations of Na and Cl. In the cells washed with 0.3 M mannitol, most of the elemental concentrations were significantly lower than in their unwashed counterparts and those washed in the other solutions. In cells washed in either 0.15 M ammonium acetate or distilled water, no significant differences in P and K compared with their unwashed counterparts were found. There were also no significant differences between cells washed in ammonium acetate and in distilled water. In subsequent experiments ammonium acetate was used. Incubation of cells in standard Ringer's solution resulted in an increase in Na and Cl, and a decrease in K, concomitantly with an increase in Ca. Although Ringer's solution per se can elicit changes in diffusible elements in the cells, physiological and pharmacological effects of oxytocin could still be detected in Ringer's solution. However, effects of oxytocin were different when the experiment was done in culture medium, instead of in Ringer's solution.

X-ray microanalysis of myometrium in parturient women at term.

The purpose of this study was to investigate the elemental composition of myometrial cells from term parturient women with normal and dysfunctional labour. Myometrial biopsies were obtained from forty-four term pregnant women undergoing Cesarean section. The patients were categorized according to uterine activity as follows: before labour, normal labour, labour successfully augmented by oxytocin, oxytocin-resistant labour, and labour arrested by terbutaline. X-ray microanalysis of freeze-dried sections (16 mu m thick) of the myometrium was carried out. An increase in intracellular phosphorus level (p < .01) was noted in the normal labour group compared to before labour. In patients with normal labour, higher phosphorus (p < .009) and potassium (p < .005) were found compared to oxytocin resistant labour. Patients with oxytocin resistant labour had lower intracellular potassium (p < .0006) and phosphorus (p < .02), and higher chloride (p < .05) and sodium (p < .03) compared to levels found in patients who responded to oxytocin treatment. In dysfunctional (oxytocin-resistant) labour the ion distribution in the myometrial cells might be disturbed. The reduced level of potassium and phosphorus together with the high sodium and chloride levels found in patients with oxytocin resistant labour may be connected to an impairment in sodium-potassium pump and muscle dysfunction, clinically diagnosed as dystocia.

The expression of 15-lipoxygenase gene and the presence of functional enzyme in cytoplasm and nuclei of pregnancy human myometria.

The expression of the 15-lipoxygenase (15-LO) gene in pregnancy human myometria, from messenger RNA (mRNA) to the product of the enzyme reaction, was investigated. In situ hybridization with antisense riboprobe synthesized from human reticulocyte 15-LO complementary DNA has revealed the presence of mRNA in myometrial smooth muscle as well as in myometrial blood vessels. Immunoblot analysis with a polyclonal antibody to recombinant human 15-LO enzyme showed a single 110-kilodalton immunoreactive protein in myometria. Light microscope immunocytochemistry using polyclonal antibodies has demonstrated the presence of immunoreactive 15-LO protein and 15-hydroxyeicosatetraenoic acid (15-HETE), the primary product of the 15-LO pathway. While myometrial blood vessels did not show any obvious change, myometrial smooth muscle showed lower 15-LO mRNA, 15-LO immunoreactive protein, and 15-HETE at term pregnancy and during labor. Immunogold electron microscopy showed the presence of immunoreactive 15-LO in rough endoplasmic reticulum and 15-HETE in myofilaments. Quite unexpectedly, both are also present in nuclear chromatin. In summary, the present study demonstrates for the first time the expression of 15-LO gene in pregnancy human myometria and the mRNA and catalytically active enzyme are lower at term pregnancy and during labor. Quite unexpectedly, catalytically active 15-LO is also present in nuclear chromatin. These findings suggest that 15-LO/15-HETE may have genomic as well as nongenomic actions, both of which may either initiate and/or facilitate the progression of labor in women.