The discovery of piezoelectric potentials in loaded bone was instrumental in developing a plausible mechanism by which functional activity could intrinsically influence the tissue's cellular environment and thus affect skeletal mass and morphology. Using an in vivo model of osteopenia, we have demonstrated that the bone resorption that normally parallels disuse can be prevented or even reversed by the exogenous induction of electric fields. Importantly, the manner of the response (i.e., formation, turnover, resorption) is exceedingly sensitive to subtle changes in electric field parameters. Fields below 10 muV/cm, when induced at frequencies between 50 and 150 Hz for 1 h/day, were sufficient to maintain bone mass even in the absence of function. Reducing the frequency to 15 Hz made the field extremely osteogenic. Indeed, this frequency-specific sinusoidal field initiated more new bone formation than a more complex pulsed electromagnetic field (PEMF), though inducing only 0. 1 % of the electrical energy of the PEMF. The frequencies and field intensities most effective in the exogenous stimulation of bone formation are similar to those produced by normal functional activity. This lends strong support to the hypothesis that endogenous electric fields serve as a critical regulatory factor in both bone modeling and remodeling processes. Delineation of the field parameters most effective in retaining or promoting bone mass will accelerate the development of electricity as a unique and site-specific prophylaxis for osteopenia. Because fields of these frequencies and intensities are indigenous to bone tissue, it further suggests that such exogenous treatment can promote bone quantity and quality with minimal risk or consequence.

We have previously shown that 1,25-dihydroxyvitamin D [1,25-(OH)2D3] and glucocorticoid modulate adenylate cyclase activation by PTH in osteoblast-like cells. Here we examine whether steroid effects on PTH receptor density explain the modulation of PTH action. Receptor assays were performed on late logarithmic-phase monolayers of ROS 17/2.8 cells using human PTH-like peptide (hPLP) as radioligand. K(d) and receptor density were computed from competition of tracer amounts of [I-125-Tyr36]hPLP-(1-36) with unlabeled hPLP-(1-36) (0.1-30 nM). Steroid treatment had little or no effect on affinity for ligand. Pretreating cells with 10 nM 1,25-(OH)2D3 for 48 h decreased PTH receptor number to 17% of control values. Treating cells with 10 nM of the glucocorticoid triamcinolone acetonide (TRM) increased receptor number 10-fold, but simultaneous treatment with 1,25-(OH)2D3 (10 nM) completely prevented this receptor increase. Steroid effects required 13-18 h of treatment. Dose-response relationships for steroid modulation, determined from binding at 0.17 nM radioligand, indicated an EC50 of 0.3 nM for glucocorticoid augmentation of PTH receptor number and 0.02 nM for 1,25-(OH)2D3 reduction of receptor number in the presence or absence of the maximum TRM effect. The initial rate of cAMP production by receptor-saturating concentrations of PTH was 11,500 molecules per receptor per minute in untreated cells, comparable to reported turnover numbers for mammalian adenylate cyclase. Control experiments were validated measuring cAMP in intact cells as an indicator of adenylate cyclase activity. Cyclic AMP production was reduced 63% by 1,25-(OH)2D3 (10 nM) treatment. GlucoCorticoid (10 nM) enhanced cAMP production twofold but reduced cAMP generation per receptor by 80%. We conclude that 1,25-(OH)2D3 reduction of PTH receptor number modulates ROS cell responses to PTH and prevents glucocorticoid-enhanced PTH action. The data also suggest that other components of the adenylate cyclase complex limit amplification of the PTH signal when PTH receptor number is elevated by glucocorticoid treatment.

U937 cells can be induced to express receptor for complement 5a (C5aR) by sequential 2 day treatments of cells with dihydroxyvitamin D-3 (1,25(OH)2D3) followed by prostaglandin E2. We asked whether the action of prostaglandin E2 to cause maximal C5aR expression required only activation of the cAMP-dependent protein kinase (PKA). Prostaglandin E2 dose dependently activated PKA in control and 1,25(OH)2D3 treated cells; by 4 h the PKA did not respond to further prostaglandin E2 challenge. We hypothesized that prostaglandin E2 actions transduced via PKA should be complete by 4 h; i.e., C5aR induction should be equivalent in cells treated with prostaglandin E2 for 4 h and for 2 days. All cells were treated for the first 2 days with 1,25(OH)2D3 and the second 2 days with prostaglandin E2 or cAMP analogs. C5aR number was measured after 4 days total culture. 4 h pulse treatments with agents were given at the end of the 1,25(OH)2D3 treatment. Cells exposed to a 4 h pulse of prostaglandin E2 had only 68.2 ± 4.4% the amount of C5aR seen in cells continuously exposed to prostaglandin E2. Continuous culture with a cAMP analog pair (50 μM each of 8-thiomethyl-cAMP + N6-benzoyl-cAMP), which caused a 41.7% ± 10.8% increase PKA activation above basal, resulted in only 51% ± 16% of the C5aR numbers seen in cells cultured for 2 days with prostaglandin E2, where PKA remained at basal activity. We therefore concluded that C5aR expression caused by prostaglandin E2 could not be ascribed entirely to duration or degree of activation of cAMP-dependent signalling pathways. We investigated the possibility that the calcium sensitive protein kinase C was involved. Cytoplasmic protein kinase C was increased 154% ± 14% above control in cells treated with sequential 2 days treatments of 1,25(OH)2D3 and prostaglandin E2. A 147% ± 2% increase in membrane associated protein kinase C was also seen 10 min after phorbol myristate acetate stimulation in the above treatment group. Finally, phorbol myristate acetate augmented the C5aR induction caused by cAMP analog. We propose that the mechanism of prostaglandin E2 synergism with 1,25(OH)2D3 in causing C5aR induction in U937 cells includes signal transduction not only by the cAMP cascade, but also via protein kinase C modulated pathways.

Glucocorticoid increases and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] decreases PTH activation of adenylate cyclase and cAMP-dependent protein kinase in rat osteosarcoma cells (ROS 17/2.8). Since selective cAMP-dependent protein kinase isoenzyme activation may account for specific physiological hormonal responses, we investigated steroid effects on activation of isoenzymes I and II in response to PTH using a new ion exchange separation procedure. Pretreatment of cells for 2 days with the glucocorticoid triamcinolone acetonide (TRM) or 1,25-(OH)2D3 altered the degree of cAMP-dependent protein kinase isoenzyme activation by PTH in accordance with their modulation of intracellular cAMP accumulation, but did not alter the amount of each isoenzyme present or the order in which isoenzymes I and II were activated. In all treatment groups isoenzyme I was preferentially activated by low doses of PTH, while high concentrations activated both isoenzymes, as predicted by the relative affinities of each isoenzyme for cAMP. Glucocorticoid reduced the concentration of bovine PTH-(1-34) required for maximal activation of isoenzyme I from 1 to 0.05 ng/ml and that required for activation of isoenzyme II from 10 to 1 ng/ml. This effect was abolished by simultaneous treatment of cells with 1,25-(OH)2D3. At doses of PTH that caused partial activation (0.05-0.1 ng/ml for isoenzyme I; 1 ng/ml for isoenzyme II), 1,25-(OH)2D3 treatment attenuated this activation. In all groups both isoenzymes were fully activated by 100 ng/ml PTH. Control experiments demonstrated that isoenzyme activation is not a result of cell disruption over the range of PTH doses that regulation by steroid hormone was observed. These results extend our studies on modulation of the cAMP pathway by steroid hormones and make it feasible to correlate selective isoenzyme activation with specific responses to PTH.

Glucocorticoids increase and 1,25-dihydoxyvitamin D3 [1,25-(OH)2D3] decreases the activity of PTH-responsive adenylate cyclase, altering intracellular cAMP in a rat osteoblast-like cell line (ROS 17/2.8). This study was undertaken to measure the subsequent activation of the cAMP-dependent protein kinase (PKA). Pretreatment of ROS cells for 2 days with the glucocorticoid triamcinolone acetonide (TRM), shifted the dose-response curve for PKA activation by PTH upward compared to the control value. Basal PKA activity was enhanced 50% by TRM, and the PTH concentration required for maximal activation of PKA decreased from 1.0 to 0.05 ng/ml. At the lowest effective PTH concentration (0.05 ng/ml) the mean PKA activity ratio increased to 0.73 in TRM-treated cells compared with 0.45 in untreated cells. Pretreatment with 1,25-(OH)2D3 had opposite effects, shifting the dose-response curve for PKA activation by PTH downward and to the right, decreasing the basal activity ratio from 0.26 to 0.16, and increasing the PTH concentration required for maximal activation to 10 ng/ml. 1,25-(OH)2D3-treated cells stimulated with 0.5-1 ng/ml PTH consistently had lower PKA activity ratios than untreated cells. Simultaneous treatment with 1,25-(OH)2D3 reversed the effect of TRM. There were no differences in total PKA activity (2.57 +/- 0.09 pmol 32P/min.micrograms protein) between treatment groups, suggesting that TRM and 1,25-(OH)2D3 do not alter the cellular PKA concentration. In control experiments exogenous PKA was added to sonication buffer of PTH-stimulated cells to verify that the TRM and 1,25-(OH)2D3 shifts in PKA activation at low PTH doses occur before sonication. cAMP-dependent protein kinase activation was also studied by measuring the progressive occupation of regulatory subunit-binding sites by hormonally stimulated endogenous cAMP. [3H] cAMP binding was expressed as the percent change in bound [3H]cAMP per microgram protein compared to that in unstimulated cells not steroid treated. [3H]cAMP binding to all cytosol fractions decreased as PTH increased over the concentration range predicted by our PKA activation experiments. TRM treatment shifted the curve for [3H]cAMP binding to regulatory subunit downward and to the left, and 1,25-(OH)2D3 treatment shifted it upward and to the right. In cells treated with both TRM and 1,25-(OH)2D3, the curve was similar to control curve. Sonicating unstimulated cells in buffer containing comparable concentrations of added cAMP did not alter [3H]cAMP binding. These and the previous controls suggest that changes in PKA activation at low doses of PKA reflect cellular events occurring before cell disruption.(ABSTRACT TRUNCATED AT 400 WORDS)

cAMP and 1,25-dihydroxyvitamin D3 [1,25- (OH)2D3] have both been shown to affect cell proliferation and phenotype. Since 1,25-(OH)2D3 modulates cellular cAMP production in the U937 cell line, we have investigated the possibility that 1,25-(OH)2D3 may interact with cAMP to affect distal cellular events. The U937 cell normally contains less than 2000 receptors/cell for the chemotactic factor C5a, as measured by concentration-dependent binding assays with the ligand probe [125I]iodo-C5a; this number rises when the cell is exposed to agents that promote cellular differentiation, such as (Bu)2cAMP. U937 cells incubated with 1,25-(OH)2D3 or cAMP agonists [prostaglandin (0.03–3μm), isoproterenol (10−7–10−5m), and forskolin (5–15 μm)] alone demonstrate no increase in C5a receptor number. When the cAMP agonists are combined with 1,25-(OH)2D3 for up to 4 days, receptors are induced to levels comparable to those achieved with pharmacological doses of (Bu)2cAMP (25,000–75,000/cell), with a similar dose inducing half-maximal binding of C5a (∼l–3 nm). The synergistic effects of 1,25-(OH)2D3 and cAMP agonists are dose and time dependent; other metabolites of vitamin D are not effective. The data suggest that 1,25-(OH)2D3 may facilitate the effects of endogenous cAMP to promote certain types of differentiation. (Endocrinology118: 2540–2545, 1986)

Silicon levels in plasma and urine have been studied in normal subjects, in chronic renal failure patients, and in regular hemodialysis patients. Plasma levels were elevated in chronic renal failure, and in hemodialysis patients they were three times the control values. Urine silicon excretion is significantly related to creatinine clearance, and to urinary omolality, magnesium and calcium levels. A small amount of residual silicon is found in the hollow fiber artificial kidneys made by some manufacturers. High concentrations of silicon are found in commercial dialysate and pass down a chemical concentration gradient into the blood compartment. It is concluded that silicon is sequestered rapidly in the body during hemodialysis, and that this amounts to 58 g of silicic acid per annum from hemodialysis alone.