Differential effects of glucocorticoids on bone resorption in neonatal mouse calvariae stimulated by peptide and steroid-like hormones

Differential effects on in vitro bone resorption were observed when the glucocorticoids, hydrocortisone and dexamethasone, were added to neonatal mouse calvariae treated with either parathyroid hormone (PTH), 1,25(OH)2-vitamin D3, all trans-retinoic acid (t-RA), or prostaglandin E2 (PGE2). Bone resorption was assessed by analyzing either the release of 45Ca from [45Ca]CaCl2 prelabeled calvarial bones or the release of 3H from [3H]proline prelabeled calvariae. At PGE2 concentrations of 3 x 10(-8) and 3 x 10(-7) mol/l, co-treatment with either 10(-6) mol/l dexamethasone or 10(-6) mol/l hydrocortisone caused additive 45Ca release from neonatal mouse calvariae. In contrast, synergistic release from mouse calvarial bones of both 45Ca and 3H was found after either 10(-6) mol/l hydrocortisone or 10(-6) mol/l dexamethasone was combined with 3 x 10(-11) mol/l PTH treatment for 120 h. Dose-response studies indicated that the synergistic stimulation of 45Ca release from neonatal mouse calvariae by glucocorticoids and PTH could be elicited at glucocorticoid concentrations of 10(-8) to 10(-6) mol/l and at PTH concentrations of 10(-11) to 10(-9) mol/l. Progesterone and RU 38486 (a derivative of 19-nortestosterone with antiglucocorticoid activity) blocked the synergism noted with glucocorticoid and PTH co-treatment, suggesting that interaction between the steroids and PTH was dependent on glucocorticoid receptor interaction. Addition of either 10(-6) mol/l hydrocortisone or 10(-6) mol/l dexamethasone to neonatal mouse calvariae treated with 1,25(OH)2-vitamin D3 (10(-11) and 10(-10) mol/l) also resulted in synergistic stimulation of 45Ca release. In contrast to these observations, the stimulatory effect of t-RA (10(-8) mol/l) on 45Ca release from calvarial bones was abolished in the presence of 10(-6) mol/l dexamethasone. These results suggest that an important role of glucocorticoids may be to synergistically potentiate bone resorption stimulated by PTH and 1,25(OH)2-vitamin D3, but indicate an opposing interaction between the glucocorticoids and bone resorptive retinoids.

The effects of low molecular weight and standard heparin on calcium loss from fetal rat calvaria

Osteoporosis is a well-recognized complication of long-term heparin use. However, the mechanisms by which heparin can influence bone metabolism are unclear. We report here that unfractionated heparin stimulates the process of bone resorption and that the low molecular weight heparins (LMWHs), enoxaparin, fragmin, logiparin, and ardeparin produce significantly less calcium loss than unfractionated heparin. To assess calcium loss from bone, we quantified the release of 45Ca into the culture medium of fetal rat calvaria. 45Ca release was increased in a dose-dependent manner by the addition of either unfractionated heparin or the LMWHs; but more than 50-fold higher LMWH concentrations were required to obtain an equivalent effect to unfractionated heparin. Thus, at concentration > or = 2 micrograms/mL (0.35 anti-Xa units/mL), unfractionated heparin stimulated 45Ca release 1.53 +/- 0.06 fold. 45Ca release was increased to a similar extent by the addition of either 10(- 7) mol/L parathyroid hormone (PTH) or 10(-6) mol/L 1,25 dihydroxyvitamin D3 (1,25 Vit D3). In contrast to unfractionated heparin, LMWH concentrations > or = 100 micrograms/mL (> or = 14.0 anti- Xa units/mL) were required before maximum isotope release was observed. At concentrations well above therapeutic levels, the LMWHs stimulated 45Ca release by only 1.25 /+- 0.01-fold. Heparins with high and low antithrombin III affinities stimulated 45Ca release equally well. Both size and sulfation were found to be major determinants of heparin's ability to promote isotope release. Thus, the ability of defined heparin fragments to stimulate 45Ca release correlated with their molecular weight, and after N-desulfation the ability of heparin to induce isotope release was greatly diminished. Dermatan sulfate had no effect on 45Ca release. We conclude that size and sulfation are major determinants of heparin's ability to promote bone resorption and that the risk of heparin-induced osteoporosis may be reduced by the use of LMWH preparations.

Compensatory downregulation of myocardial Ca channel in SR from dogs with heart failure

In this study we tested the hypothesis that the ryanodine-binding Ca-release channel activity and density of the sarcoplasmic reticulum (SR) terminal cisternae were decreased in congestive heart failure (CHF) that occurs spontaneously in doberman pinschers or experimentally with rapid ventricular pacing of mongrels. We used a novel, sensitive, and easy-to-perform microassay and demonstrated a 50% decrease in activity of the myocardial SR Ca pump and a 75% reduction in SR Ca-release channel activity in CHF. Decreases in Ca channel content were associated with increases in net Ca sequestration. 45Ca-release experiments from passively loaded SR terminal cisternae and ryanodine-binding studies confirmed a 53–68% downregulation of the Ca-release channel activity. As a consequence of release channel downregulation, there was partial restoration of net Ca sequestration activity in dogs with CHF and complete compensation in dogs with mild cardiac dysfunction. Deterioration of Ca cycling correlated with deterioration of myocardial performance, apparently due to decreased Ca-adenosinetriphosphatase (ATPase) pump and not Ca channel content. One-half the reduction in Ca-release activity could be attributed to decreased Ca sequestration and one-half to decreased Ca channel density. Downregulation of Ca channel content decreases the amplitude of the Ca cycle and maximizes the downregulation of Ca pumps that may occur. Although these adaptations may reduce cellular energy expenditure, they are likely to render the myocardium more susceptible to fatigue and failure.

Greater unidirectional calcium efflux from bone during metabolic, compared with respiratory, acidosis

There is a smaller net calcium efflux from bone in vitro during respiratory (increased PCO2) than metabolic (decreased [HCO3-] acidosis. This could be due to the elevated PCO2, which would lessen the driving force for mineral dissolution and increase the driving force for mineralization with respect to carbonated apatite in the bone mineral. To test this hypothesis, we injected neonatal mice with 45Ca and dissected the radiolabeled calvariae 24 h later. The live calvariae were then cultured for 24 h under conditions simulating respiratory acidosis (Resp, pH = 7.225 +/- 0.003, PCO2 = 87.5 +/- 0.1 mmHg), severe respiratory acidosis (SResp, pH = 7.072 +/- 0.004, PCO2 = 103.0 +/- 0.5 mmHg), metabolic acidosis (Met, pH = 7.212 +/- 0.003, HCO3- = 15.5 +/- 0.1 meq/l), or normal acid-base status (Ctl, pH = 7.452 +/- 0.003, PCO2 = 40.0 +/- 0.2 mmHg, HCO3- = 27.8 +/- 0.2 meq/l) and bidirectional net calcium flux (JCa) and unidirectional 45Ca release were determined. There was greater JCa from bone during Met than Resp, and JCa was not different from Met during SResp despite the latter having a significantly lower pH. There was greater unidirectional 45Ca release from bone during Met than Resp, SResp, or Ctl. There was a similar direct correlation between JCa and 45Ca efflux in the respiratory and metabolic groups. However, when calvarial osteoclast activity was inhibited with calcitonin,although there was again greater JCa and 45Ca release with a metabolic compared with respiratory acidosis, there was a greater proportion of 45Ca release than JCa from bone.(ABSTRACT TRUNCATED AT 250 WORDS)

Ca exchange under non-perfusion-limited conditions in rat ventricular cells: identification of subcellular compartments

Freshly prepared ventricular myocytes from rat hearts, aliquots of which were tested for sarcolemmal integrity by La exposure, were labeled at high 45Ca specific activity. Isotope was subsequently washed out at a perfusion rate of 2.8 ml/s with washout solution sampled each 1 s. No initial unrecorded period of washout was imposed. Four compartments were distinguishable: 1) a "rapid" compartment (RC) containing 2.6 mmol Ca/kg dry wt of La-displaceable Ca, half time (t1/2) less than 1 s; 2) an "intermediate" compartment(s) (IC) containing 2.1 mmol, t1/2 = 3 and 19 s; 3) a "slow" compartment (SC) containing 1.6 mmol, t1/2 = 3.6 min; 4) an "inexchangeable" compartment that demonstrated no 45Ca uptake after 60-min labeling containing 1.2 mmol. Introduction of 10 mM caffeine as a probe for sarcoplasmic reticulum (SR) content at various times during the washouts caused an increased release of 45Ca. The net increased 45Ca release plotted as a function of time at which caffeine was introduced produced a biexponential curve with t1/2s of 2 and 22 s, very similar to the t1/2s of the IC. Ryanodine (1 microM) significantly reduced the caffeine-induced 45Ca release, confirming the SR locus of the IC. Cells were perfused with 10 mM NaH2PO4 to specifically increase mitochondrial 45Ca labeling. Subsequent removal of PO4 at various times during washouts produced large increases in effluent 45Ca. A plot of the net peak release of 45Ca vs. time of PO4 removal was monoexponential with t1/2 = 3.3 min, very similar to the SC t1/2. The large La-accessible RC remains unlocalized, but the rapidity of its exchange places it in the sarcolemma and/or at sites in rapid equilibrium with the sarcolemma.

Adenylate cyclase blockers dissociate PTH-stimulated bone resorption from cAMP production

It is uncertain whether adenosine 3',5'-cyclic monophosphate (cAMP) or the inositol-calcium pathway mediates the stimulation of bone resorption by parathyroid hormone (PTH). Incubation of bone organ cultures with cAMP analogues and forskolin has not resolved this question because of the cellular inhomogeneity of bone and the consequent presence of adenylate cyclase-linked receptors for both PTH and calcitonin, hormones with opposite effects on bone resorption. We have used two new inhibitors of adenylate cyclase, 9-(tetrahydro-2-furyl)adenine (SQ 22536) and 2',5'-dideoxyadenosine (DDA), to directly reassess the role of cAMP in PTH-stimulated osteolysis. SQ 22536 (0.01-1.0 mM) and DDA (0.01-1.0 mM) completely blocked PTH stimulation of cAMP production measured in the absence of a phosphodiesterase blocker. In the presence of 1 mM 3-isobutyl-1-methylxanthine, half-maximal inhibition of PTH-induced cAMP production occurred with 0.2 mM SQ and 0.1 mM DDA, respectively. These concentrations of SQ and DDA had no effect on PTH-stimulated 45Ca release from calvaria, although both agents inhibited bone resorption when present at concentrations of 1-2 mM. At these levels, SQ and DDA caused equivalent inhibition of 45Ca release stimulated by 1,25-dihydroxyvitamin D3 but did not affect basal 45Ca release or [3H]-phenylalanine incorporation. It is concluded that substantial blockade of PTH-induced cAMP production does not affect this hormone's stimulation of bone resorption, which is therefore likely to be mediated by another intracellular messenger system, possibly calcium. In millimolar concentrations, SQ and DDA appear to be nonspecific blockers of osteoclastic bone resorption.

Forskolin and caffeine induce Ca2+ release from intracellular stores in rabbit aorta

We combined techniques of 45Ca efflux and computer-assisted kinetic analysis to investigate the effects of forskolin and caffeine on intracellular Ca2+ metabolism in intact rabbit aortic segments. When either 1 microM forskolin or 10 mM caffeine was present during the 45Ca load and efflux, the amount of 45Ca released from the tissue was reduced during the interval between 50 and 250 min of efflux. In contrast caffeine but not forskolin induced an acute increase in 45Ca efflux when added to the perfusion medium after 1 h of efflux in physiological salt solution. Preincubation with caffeine abolished phenylephrine-stimulated 45Ca release, but preincubation with forskolin had no effect. Kinetic analysis of these data indicated that caffeine reduced the Ca2+ content of the same intracellular compartment depleted by phenylephrine, whereas forskolin depleted a different intracellular Ca2+ store. Forskolin-induced depletion of an intracellular Ca2+ store was surprising in light of current evidence suggesting adenosine 3',5'-cyclic monophosphate stimulation of sarcoplasmic reticular Ca2+ uptake, but hypotheses that included only this mechanism are inconsistent with our findings.