Membrane signalling and progesterone in female and male osteoblasts. I. Involvement Of intracellular Ca(2+), inositol trisphosphate, and diacylglycerol, but not cAMP.

Bone is a target tissue of progestins, but the mechanisms by which they act are still unclear. We examined the early (5-60 s) effects of progesterone and progesterone covalently bound to BSA (P-CMO BSA), which does not enter the cell, on the cytosolic free Ca(2+) concentration ([Ca(2+)]i) and the formation of inositol 1,4,5 trisphosphate (InsP3) and diacylglycerol (DAG) in confluent female and male rat osteoblasts. P-CMO BSA like free progesterone increased [Ca(2+)]i via Ca(2+) influx through L-type Ca(2+) channels and Ca(2+) mobilization from the endoplasmic reticulum. Both progestins increased InsP(3) and DAG formation within 10 s, and the increase was blocked by phospholipase C inhibitors (neomycin and U-73122). Progesterone and P-CMO BSA mobilized calcium from the endoplasmic reticulum via the activation of a phospholipase C linked to a pertussis toxin-insensitive G-protein in both osteoblast types, and this process was controlled by protein kinase C. Neither progestin had any effect on cAMP formation in male and female osteoblasts. The membrane effects were not blocked by a progesterone nuclear antagonist. They were independent of the concentration of nuclear receptors and not linked to gender. Thus, progesterone appears to act in female and male rat osteoblasts via unconventional cell-surface receptors which belong to the class of membrane receptors coupled to phospholipase C via a pertussis toxin-insensitive G-protein. The bifurcating pathways leading to the formation of InsP(3) and DAG may provide a certain flexibility in controlling cell responses, both by their nature and by their rates of formation and degradation.

Cell signaling and estrogens in female rat osteoblasts: a possible involvement of unconventional nonnuclear receptors.

Estrogen deficiency is associated with bone loss, and estrogen replacement is an effective treatment of this osteoporotic process. This study examines the early (5-120 s) effects of 17 beta-estradiol on the intracellular calcium and phospholipid metabolism in confluent female rat osteoblasts. The cytosolic free Ca2+ concentration ([Ca2+]i) was determined using fura-2/AM as Ca2+ probe. Cells were labeled with myo-[2-3H]inositol or [14C]arachidonic acid for inositol or lipid determination. Inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) production were determined by either mass measurement or anion-exchange chromatography or by thin-layer chromatography, respectively. 17 beta-Estradiol (1 pM to 1 nM) increased [Ca2+]i in a biphasic manner within 10 s via Ca2+ influx from the extracellular milieu, as shown by the effects of the calcium chelator EGTA and the Ca2+ channel blockers nifedipine and verapamil, and via Ca2+ mobilization from the endoplasmic reticulum (ER), as shown by the effects of thapsigargin. 17 beta-Estradiol (1 pM to 1 nM) induced a biphasic and concomitant increase in IP3 and DAG formation. Estradiol immobilized on bovine serum albumin (BSA) [E-(O-carboxymethyl)oxime BSA] and its derivative (O-carboxymethyl)oxime rapidly increased ([Ca2+]i, IP3, and DAG and were full agonists, although they were less potent than the free estradiol. They had the same action time course and acted via Ca2+ influx and Ca2+ mobilization from ER. Tamoxifen, a potent inhibitor of genomic steroid responses, did not block the rapid increase in Ca2+, IP3, and DAG induced by estradiol. Finally, inhibitor of phospholipase C (neomycin) and pertussis toxin abolished the effects of 17 beta-estradiol on IP3 and DAG formation. These results suggest that female rat osteoblasts bear non-genomic unconventional cell surface receptors for estradiol, belonging to the class of the membrane receptors coupled to a phospholipase C via a pertussis toxin-sensitive G protein.

Uterine cells other than stromal decidual cells are required for 1,25-dihydroxyvitamin D3 production during early human pregnancy.

Human decidual cells are known to produce 1,25-(OH)2D3 at the end of pregnancy, the present study evaluates this capacity, and the part played by stromal decidual cells, in early pregnancy. Cells were obtained from nine human decidua by aspiration or curettage during early pregnancy (7-10 weeks), separated on Ficoll-Paque and plastic adherence, and incubated for 1 h with 25-(OH)D3. Incubation medium and cells were extracted and chromatographed on two successive HPLC systems. The cells examined were of both physiological and pathological (ectopic pregnancy) origin. Endometrial cells obtained in four non-pregnant situations (myomas) were also studied to determine whether the 1,25-(OH)2D3 synthesis by the uterus is associated with the appearance of decidual cells. Results show that human decidual cells from early pregnancy convert 25(OH)D3 (2.5 nM or 2.5 microM) into a metabolite with the physicochemical characteristics of synthetic 1,25-(OH)2D3. This ability is shared by cells isolated during early pregnancy, whether physiological or ectopic (tubal pregnancy). Non-adherent cells, which include mainly stromal decidual cells, are less able to produce 1,25-(OH)2D3 than are the adherent cells, suggesting that macrophages, granulocytes or as yet unidentified cell types are required for the 1,25-(OH)2D3 production by decidual tissue during early human pregnancy. In addition, one out of four experiments with non-pregnant endometrial cells could produce 1,25-(OH)2D3 suggesting that, although not the rule in the non-pregnant state, in vitro production of 1,25-(OH)2D3 by uterine cells can be found in the absence of decidual cells.

Hormonal and local factors control the immunohistochemical distribution of immunocytes in the rat uterus before conceptus implantation: effects of ovariectomy, fallopian tube section, and injection.

The distribution of immunocytes in the rat uterus undergoes profound changes during early pregnancy. This study was designed to evaluate the respective contributions of hormonal and local factors to regulation of the distribution and number of MCA341+ monocyte-macrophage antigen-bearing cells and T-lymphocyte-polymorphonuclear leukocyte (PMN) antigen-bearing cells before and during implantation of the fertilized ovum. Immunohistological data in normal rat pregnancy were compared to those found in cycling rats, ovariectomized rats, pseudopregnant rats (the oviducts of which had been sectioned on Day 0.5 of pregnancy), and pregnant rats injected with the antiprogesterone RU-486 on Day 0.5 of pregnancy. Four major events were observed: (1) transient accumulation of T-lymphocyte-PMN antigen-bearing cells in the endometrium close to the lumen and occurring only in the pregnant state 12 h after mating; (2) accumulation of an MCA341+ antigen-bearing monocyte-macrophage subset in the uterus, especially the luminal endometrium, 12 h after ovulation in pregnant as well as cycling rats; (3) progressive disappearance of these labeled cells starting 1 day after ovulation in the pregnant and nonpregnant states and influenced by RU-486 injection; (4) relative persistence of labeled cells in the deep endometrium before the implantation of the conceptus--which requires the presence of fertilized ovum in the genital tract. In conclusion, a complex multifactorial and sequential control of the distribution and number of cells bearing MCA341+ monocyte-macrophage or T-lymphocyte antigens appears to be at work before and during implantation of the rat conceptus, and may involve hormonal factors as well as local factors produced by the embryo or trophoblastic cells.