formation in osteoblasts:

Stimulation of phosphorylation pathways in ROS 17/2.8 cells mimics the effect of 1,25D on ion channel activities. There is previous evidence for modulation of L-Ca channels in osteoblasts by cAMP associated with actions of the natural vitamin D metabolite 24,25(OH)2-vitamin D3 and parathyroid hormone. We found that the cell permeant cAMP analog diBucAMP poten­tiates (about 2.5-fold at 0 mV) inward Ca2+ currents after 5 min in a fashion clearly similar to hormone 1,25D, and causes a signifi­cant shift in current to voltage relations to negative values. This shift facilitates the opening of L-Ca channels at membrane potentials close to the resting value, and promotes the influx of Ca2+ ions from the extracellular medium.

Table I – Summary of 1,25D effects on voltage-gated Ca2+ and Cl- channels in osteoblasts. V: voltage; PTX: Pertussis toxin.

CHANNEL TYPE

L-Ca

Cl

1,25D EFFECTS

Shifts V-sensitivity (seconds)

Increases conductance (minutes)

1,25D MODE OF ACTION

Direct interaction and signal transduction from a
membrane-associated receptor

Signal transduction from a membrane-asso­ciated receptor coupled to
a PTX-insensitive Gq protein

cAMP

cAMP (40% of cells)

Phosphorylation

Phosphorylation (PK)

VDR is necessary

VDR is necessary

In addition, we found that forskolin (20 mM), an adenylate cy- clase activator, causes a significant Cl current increase in 40% of ROS 17/2.8 cells after 2 min. Pre-incubation of cells with 0.05 mM staurosporine, a broad spectrum PK inactivator, in­hibited 1,25D-potentiation of Cl currents. Taken together, these results indicate that 1,25D appears to potentiate Clchannel functions in ROS 17/2.8 cells through phosphorylation of the channel molecule via a cAMP/PKA pathway. It has been observed that, in general terms, steroids affect ion channel activities through different mechanisms. Most studies have shown steroid blocking effects at micromolar concentra­tions. Our work in osteoblasts, however, uses nanomo­lar, physiological concentrations of 1,25D. The hormone modu­lates the voltage-sensitivity of L-Ca channels by shifting current to voltage relations to more negative potentials, on one hand, and increases the conductance of voltage-gated Clchannels, on the other. Our observations therefore reveal multiple mechanisms of action of 1,25D on ion channel activities that take place simultaneously in the osteoblast plas­ma membrane. Table I summarizes 1,25D effects on ion chan­nel types in ROS 17/2.8 cells, and the mechanisms of action that we propose [extracted from].

Figure 4 - 1,25D potentiation of Cl

Figure 4 – 1,25D potentiation of Cl- currents in VDR WT but not in KO osteoblasts. Fold increase (average ± S.D.) of whole-cell outward Cl- currents promoted by the addition of 5 nM 1,25D. Current amplitudes were measured at -80 mV from genotyped VDR +/+ (n = 6), +/- (n = 6), and -/- (n = 11) mouse calvarial osteoblasts. [Duplicated with permission from].

We recently reported that the VDR is necessary for rapid 1,25D-potentiation of Cl and Ca2+ currents in osteoblasts. Treatment with 5 nM 1,25D remarkably increased Cl currents within 1-5 min in cells expressing a functional VDR, but not in osteoblasts isolated from a VDR KO mouse (see Figure 4). In addition, the hormone did not alter the voltage-sensitivity of Ca2+ channels in VDR KO osteoblasts. To carry out these studies, we used primary calvarial osteoblasts isolated from neonatal VDR KO mice. We used the Tokyo VDR KO mice generated by targeted ablation of exon 2. VDR wild type (WT) and knockout (KO) mice had been used previously to characterize the physiological status caused by an abolished vitamin D metabolism. We detected the presence of the VDR molecule by immunocytochemistry in ROS 17/2.8 cells with the use of a primary antibody against VDR and a FITC-conjugated anti-mouse secondary antibody. Figure 5 shows that the VDR localizes abundantly in the cytoplasm and in the proximity to the plasma membrane.
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Figure 5 - Subcellular localization

Figure 5 – Subcellular localization of the VDR in ROS 17/2.8 cells. This confocal immunofluorescence micrograph shows a strong signal in the cell cytoplasm and portions of the plasma membrane region (arrow). Bar = 40 jM.