The study of membrane-initiated, non-genomic actions of steroids in different cell systems has become a rapidly expand­ing field of research in recent years. The idea of steroids exert­ing a rapid effect at the cell membrane level was first formulat­ed by Szego and collaborators several decades ago. However, the majority of our current knowledge on steroid mechanisms of action resides mainly at the genomic level, with relatively less research addressing rapid or non-genomic stud­ies. Among different functions attributed to non-genomic ac­tions of steroids are those that involve rapid ion movements across the cell membrane and rapid membrane fusion as part of secretory processes in different tissues involving cytoplas­mic signaling pathways. In osteoblasts, however, there is no current consensus on how the non-genomic 1,25D signal­ing cascade is initiated. Some groups propose that the VDR or a slightly modified protein is responsible for initiating rapid re­sponses; others propose the existence of a membrane-as­sociated receptor of different molecular structure. It has been shown that hormone 1,25D rapidly increases intra­cellular Ca2+ in confluent mouse osteoblasts; 70% of this response comes from external Ca2+ entering through Ca2+ channels in the plasma membrane, and 30% from Ca2+ re­leased from the ER. In osteosarcoma ROS 17/2.8 cells, low doses (0.1-1 nM) of 1,25D promote an acute, transient (1 min long) rise in intracellular Ca2+ concentration in 40% of the cells that is entirely dependent on extracellular Ca2+ (it is blocked by Ca2+ channel blockers such as dihydropyridines). At higher doses (over 1 nM), 1,25D-promotes an increase in intracellular Ca2+ due to both the influx of extracellular Ca2+ and its release from intracellular stores. Figure 1 shows the increase in cytoplasmic Ca2+ concentration in a single live ROS 17/2.8 cell 1 min after the addition of 5 nM 1,25D to the bath. The Ca2+ signal lasted approximately 1 sec, and progressed inside the cytoplasm in a wave fashion.

In addition, hormone 1,25D rapidly activates (sec-min) the phosphatidylinositol (PI) cycle leading to formation of the sec­ond messenger IP3 in mouse osteoblasts and ROS 17/2.8 cells. Local intracellular Ca2+ elevation is crucial for the fu­sion of secretory vesicles to the plasma membrane and exocy- tosis. This occurs via activation of phospholipase C (PLC)- Pi linked to a pertussis toxin (PTX)-insensitive G-protein. IP3 promotes the release of Ca2+ from intracellular stores.

Figure 1 - 1,25D stimulated cytoplasmic calcium

Figure 1 – 1,25D stimulated cytoplasmic calcium wave in a ROS 17/2.8 cell. Series of confocal microscope Ca2+ images obtained from a single cell after the addition of 5 nM 1,25D to the bath. Sequential images were obtained with a confocal fluorescence microscope every 0.33 sec.

PLCs are effectors of different subunits of various G-proteins. Several G-protein subunits (Gaq, Gas, Gai, Gp and Gy) have been identified in female rat osteoblasts. It has been known that the effect of 1,25D on IP3 formation and Ca2+ mobilization in osteoblasts involves Gaq.
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