In excitable cells, Ca2+ channels function as transducers of an electrical signal (action potential) into a chemical signal (exocy- tosis of neurotransmitters). Osteoblasts, however, are non-excitable cells, and 1,25D appears to act in lieu of action potentials to facilitate the opening of Ca2+ channels, Ca2+ influx, and an immediate exocytotic response. Recently, Cl– channels have been shown to play a crucial role in exocytosis. Located in the secretory vesicle membrane and the plasma membrane, Cl– channels provide the electrical shunt for acidification of the vesicle content by a H+-ATPase and loading of secretory proteins. In р-pancreatic cells, for example, insulin secretion is strongly inhibited by the stilbene derivative DIDS, and the ClC- 3 channel is localized in the secretory vesicle membrane. Thus, 1,25D appears to modulate osteoblast exocytosis by acting on the ion channels directly involved in the secretory process. The 1,25D-sensitive L-Ca and Cl– channels are therefore likely to be the transducers of a hormonal signal into an electric signal (local depolarization-repolarization) coupled to secretion.
Osteoblasts produce new bone by secreting a complex extracellular matrix that has the capacity to mineralize when adequate amounts of calcium and phosphate are supplied. Bone matrix proteins include type I collagen as the major component, and proteoglycans, glycoproteins and gamma-carboxylat- ed proteins. Osteoblasts express the protein components of the SNARE complex involved in the docking of secretory vesicles to the plasma membrane during regulated secretion. Regulated secretion of proteins, which is typical of neurosecre- tory, endocrine and exocrine cells, occurs as a rapid fusion of vesicles docked to the plasma membrane in response to a specific electrical or chemical stimulus. This is associated with changes in the electrical state of the plasma membrane and elevation of cytoplasmic Ca2+. On the other hand, constitutive secretion of proteins had been traditionally associated with osteoblast physiology until recently. This is a continuous process controlled at the genomic level that comprises the production, packing, shipping, and continuous release of secretory products such as matrix proteins. Our recent finding of a regulated type of secretion stimulated by a steroid in osteoblasts is a novel concept. The molecular mechanisms underlying this secretion remain to be elucidated. Figure 6A shows the presence of secretory granules docked to the plasma membrane of a single ROS 17/2.8 ostoeblast. SEM images in Figures 6B, C show collagen fiber production by secretory mechanisms in 8 day-old cultures. We found that steroid-regulated secretion occurs in osteoblasts expressing a functional VDR, but not in cells obtained from a KO VDR mouse. Hormone 1,25D, by acting rapidly at the os- teoblast plasma membrane level via interaction with a membrane-associated VDR, promotes the release within seconds- minutes of the secretory content -typically collagen- of membrane-docked vesicles. The anabolic effects of the hormone in bone can therefore be explained at both the genomic (synthesis of proteins) and non-genomic (exocytosis of proteins) levels.
Figure 6 – Images of secretory vesicles (A) and associated secretory activities (B, C) in ROS 17/2.8 cells at early stages of bone formation. A, Secretory vesicles (arrow in A) can be observed in close proximity to the plasma membrane. Cells were stained with 3 jM octadecyl rhodamine and observed with confocal fluorescence microscopy. B, C: SEM images obtained from a 4-day old ROS 17/2.8 cell culture. Cells were treated with 5 nM 1,25D for 5 min before fixation with 2.5% glutaraldehyde in Na cacodylate buffer. In B, ROS 17/2.8 cells colony. In C, detail of the plasma membrane surface of a ROS 17/2.8 cell at high magnification. Notice the presence of collagen fibers on the cell surface.
We demonstrated in osteoblasts that a rapid physiological response to 1,25D is the activation of secretion. This happens simultaneously with Cl– channel activation. We performed continuous recordings of whole-cell capacitance as a measure of exocytosis in single cells. The addition of 1,25D to the external solution increased the frequency of individual exocytotic events within the first 1-5 min only in osteoblasts isolated from VDR wild type mice. Figure 7 shows typical capacitance responses to 1,25D obtained from a single live VDR WT osteoblast.
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Figure 7 – 1,25D stimulation of exocytotic activities in osteoblasts. Whole-cell capacitance recordings obtained from a single VDR WT osteoblast in the absence (top trace) and presence (bottom trace) of 5 nM 1,25D3 added to the bath. Upward deflections from an initial capacitance value of 53.4 pF (dotted line) represent the fusion of individual secretory granules to the osteoblast plasma membrane (exocytotic events). Notice increased frequency of events obtained after hormone addition (bottom trace). [Duplicated with permission from].