Steroids affect in different ways the activity of ion channels pre­sent in the plasma membrane of target cells. Our studies showed for the first time that physiological nanomolar concen­trations of 1,25D increase outward Cl currents in primary os­teoblasts and osteosarcoma ROS 17/2.8 cells within the first 5 minutes of treatment (see Figure 2A, B). This 1,25D-sensitive Cl channel is a voltage-gated, volume-sensi­tive anionic channel that activates upon depolarization. The presence of different Cl channel types in osteoblasts has been demonstrated by means of electrophysiological studies. The 1,25D-sensitive Cl channel that we characterized in ROS 17/2.8 cells and mouse calvarial osteoblasts shares some electrical and pharmacological characteristics with a volume- sensitive Cl channel described by Gosling et al. and a cAMP-activated Cl channel found in primary osteoblasts. It opens upon depolarization and is blocked by the specific Clchannel blocker DIDS in a time and voltage-dependent man­ner.

Figure 2 - Effect of 1,25D on outward

Figure 2 – Effect of 1,25D on outward Cl- currents in ROS 17/2.8 cells.
A, Current to voltage (I/V) relations. Inward Ca2+ currents were recorded at the beginning of the experiment (closed circles). Subsequent addition of 0.05-5 nM 1,25D to the bath activated an outward Cl- current (open symbols) within 2.5 min at depolarizing potentials. Increasing concentrations of 1,25D (0.5 nM and 5 nM, open squares and triangles, respectively) promoted increasingly larger outward Cl- currents over the entire range of depolarizing potentials. Test voltages for I/V curves were 50 ms-long and ranged from 30 to 80 mV. Holding potential -70 mV.
B, Current traces corresponding to amplitude values depicted in A, for control inward (Ca2+) currents before the addition of 1,25D, and 1,25D- potentiated outward (Cl-) currents. C, Expression at the mRNA level of the voltage-gated ClC-3 channel in ROS 17/2.8 cells cultured for 4 days. For RT-PCR experiments, we used templates designed from previously cloned rat Cl- channels (cDNA sequences were obtained from GenBank). ROS 17/2.8 cells expressed the outward rectifier, DIDS-sen- sitive ClC-3, but not the ubiquitous swelling-activated ClC-2. Transcripts of the intracellular ClC-7 channel were also found, although in signifi¬cantly lower amounts.
ClC-2, ClC-3 and ClC-7 channel genes in the osteoblastic ROS 17/2.8 cell line by means of PCR amplification. As shown in Figure 2C, we found that the ClC-3 gene is expressed in the osteoblasts, which agrees with our electrical and pharmacological characterization of the 1,25D-sensitive Cl- channel. Some cation channels activate within

Chloride channels regulate a variety of physiological and cellu­lar functions, including cell volume, electrical excitability, trans­port of salts, and exocytosis. Most mammalian cells ex­press members of the ClC gene family of voltage-gated chlo­ride channels. The ClC gene family is composed of at least nine members (ClC-1 through 7, ClC-Ka and Kb) present in the plasma membrane or in the membranes of intracellular compartments. The ClC protein structure has recently been re­vealed by X-ray crystallography. To our knowledge, how­ever, the molecular identity of Cl channels expressed in os­teoblasts remains unknown. Among these is the 1,25D-sensitive Cl channel. One possible candidate is the ClC-2 gene. The ClC-2 is a broadly expressed channel that typically activates by cell swelling and hyperpolarization. It has been described in epithelial cells, and is involved in transport of ions across epithelia, and cell volume regulation. The Cl chan­nel described by Gosling et al. in ROS 17/2.4 cells acti­vates in hyposmotic conditions. The 1,25D-sensitive Cl chan­nel that we described in the same osteoblastic cells is also a volume-sensitive channel. However, 1,25D-potentiation of the currents occurs also under isotonic conditions. Another possible candidate for the 1,25D-sensitive Cl channel protein is the one encoded by the ClC-3 channel gene.

Figure 3 - Effect of 1,25D on inward

Figure 3 – Effect of 1,25D on inward Ca2+ currents in ROS 17/2.8 cells. I/V relations obtained for L-Ca channels (A) before (closed circles) and after (open symbols) the addition of 0.5 and 5 nM 1,25D to the bath. Calcium currents were blocked by 100 |jM CdCl2 added to the bath at the end of the experiment. B, Current traces corresponding to amplitude values depicted in A, for control Ca2+ currents before the addition of 1,25D, and for 1,25D-potentiated L-Ca currents. Pulse protocols are as in Figure 2 for 200 ms-long voltage pulses. [Duplicated with permission from].

This channel is also expressed broadly, and seems to be mostly associated with endosomal and synaptic membranes. It is inhibited by DIDS and shows outward rectification, and in these respects it resembles the osteoblast 1,25D-sensitive Cl chan­nel. On the other hand, there is a ClC-7 channel described in osteoclasts. Mutation of this gene has been associated with se­vere osteopetrosis in mice and humans. ClC-7 is present in vesicle membranes in the osteoclast ruffled border, and thus participates in acidic dissolution of the inorganic components of the bone. There are no reports on ClC-7 in osteoblasts so far. With these facts in mind, we searched for the expression of seconds-minutes with physiological concentrations of 1,25D in osteoblasts. The best known 1,25D-sensitive cation channel in osteoblasts is a high voltage activated calcium channel shown in Figure 3. When added to the bath, 1,25D causes a shift in the activation of Ca2+ currents to more negative membrane potentials within the first 5 sec. This is a dihydropyridine-sensitive, or L- type Ca2+ (L-Ca) channel. The precise molecular mechanisms of 1,25D effects on the osteoblast L-Ca channel remain un­known. There is evidence that voltage-dependent L-Ca chan­nels need to be phosphorylated to respond to membrane depo­larization. Protein kinases A and C, cAMP, and GTP-binding proteins are involved in the phosphorylation of Ca2+ channel subtypes. In osteoblasts, we have shown that cAMP mimicks 1,25D-promoted changes in voltage-activation of the L-Ca channel. In addition, in muscle cells, 1,25D rapidly activates L-Ca channels via a non-genomic mechanism that involves a G protein-mediated stimulation of the adenylate cyclase/cAMP/PKA messenger system.
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