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Department of Anatomy and Developmental Biology, University College London, London WC1E 6BT, UK
* To whom correspondence should be addressed. E-mail: t.arnett{at}ucl.ac.uk.
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ABSTRACT |
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B ligand. Osteoclasts may respond to pH changes via H+-sensing ion channels such as transient receptor potential vanilloid 1, a nociceptor that is also activated by capsaicin. Acidosis also exerts a powerful, reciprocal inhibitory effect on the mineralization of bone matrix by cultured osteoblasts. This is caused by increased hydroxyapatite solubility at low pH, together with selective inhibition of alkaline phosphatase, which is required for mineralization. Diets or drugs that shift acid-base balance in the alkaline direction may provide useful treatments for bone loss disorders.
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Introduction |
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TOPABSTRACTIntroductionLITERATURE CITED |
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Acid-base balance
Causes of acidosis.
Blood pH is mainly buffered via the 
system. Additional buffering is contributed by the numerous histidine residues of hemoglobin and by plasma proteins. Addition of CO2 to the system as a result of respiration causes an increase in H+ concentration (i.e., pH reduction) leaving the 
concentration relatively unaltered. If insufficient CO2 is expelled via the lungs, a respiratory acidosis results. Conversely, addition of H+ to the system, for example as a result of the metabolism of sulfur-, nitrogen-, and phosphorus-containing molecules, will decrease pH and reduce levels without altering the CO2 concentration much. Protons generated in this way, together with associated waste anions, must be excreted via the kidneys to produce an acidified urine; if insufficient H+ is eliminated, a metabolic acidosis results.
A multitude of potential causes of systemic acidosis exist, in addition to renal and respiratory disease. These include anaerobic exercise, gastroenteritis, excessive consumption of protein or other acidifying substances, diabetes, anemias, AIDS, aging, and the menopause. Acidosis can also arise locally as a result of growth factor or cytokine stimulation of cell metabolism, vascular disease, ischemia, inflammation, infection, tumors, wounds, and fractures. It should be borne in mind that although the pH of arterial blood is normally close to 7.40, and that of venous blood 
7.36, the pH of the interstitial fluid film bathing cells in tissues will generally be lower and subject to complex gradients, depending on the metabolic activity of the cells, their distance from the nearest capillary, and the quality of the microvasculature. Because of obvious technical difficulties, this is not a well-investigated area. Data are not available for bone, but in normal skin, interstitial pH is 7.1 (1).
Acidosis and osteoclast function.
The negative impact of systemic acidosis on the skeleton has long been known (2,3) but was generally thought to result from physicochemical dissolution of bone mineral, i.e., that the skeleton acted as a giant ion-exchange column to buffer systemic acidosis in a passive manner (4,5). However, cell culture experiments showed that protons exert a direct stimulatory effect on resorption pit formation by rat osteoclasts cultured on bone slices (6). Similar acid-activation responses occur with avian and human osteoclasts (7,8) (Fig. 1). Osteoclastic resorption in these simple in vitro systems involves removal of both the mineral (mainly hydroxyapatite) and organic (mainly type 1 collagen) components of bone (or dentin) to create distinctive pits and trails similar to those seen in vivo. These experiments show that osteoclasts are almost inactive at pH 7.4 and that bone resorption increases steeply as pH is reduced, reaching a plateau at about pH 6.8. The sensitivity of osteoclasts to extracellular H+ is such that pH reductions of 
0.1 unit can be sufficient to cause a doubling of resorption pit formation (9). This effect is not subject to desensitization in longer-term cultures: acid-activated osteoclasts continue to form resorption pits over periods of 7 d or more, amplifying the effects of modest pH differences (8). Acidosis stimulates resorption in calvarial bone organ cultures similarly. Furthermore, H+-stimulated Ca2+ release from calvaria is almost entirely osteoclast-mediated, with only a small physicochemical component (10,11). These observations strongly suggest that the effects of acidosis on bone loss in vivo are also osteoclast mediated.
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B ligand (RANKL),4 1,25-dihydroxycholecalciferol (1,25D3) (Fig. 2), parathyroid hormone (6), and ATP/ADP (12). It is worth noting that proresorptive agents such as RANKL and parathyroid hormone have little or no stimulatory activity on osteoclasts at pH 7.4 or above. Thus, osteoclast stimulation is a 2-step process with acid activation as the key initial requirement, and extracellular protons may be regarded as the long-sought osteoclast activation factor.
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Progress has also been made toward understanding the mechanisms by which osteoclasts detect changes in extracellular pH in such a sensitive manner. Several classes of proteins could function as extracellular pH sensors within the relevant pH range. Two H+-sensing G-protein-coupled receptors, TDAG8 and OGR1, are expressed by osteoclasts and osteoblasts (16,17); however, neither receptor appears to be involved in regulating osteoclast activity (18). Several acid-sensing ion channels are also expressed by bone cells (19), although their function remains to be defined. The P2X2 receptor for extracellular ATP is also acid-activated and is expressed by osteoclasts but probably does not play an important role in the activation of resorption (12). Recent work suggests, however, that the acid-activated TRPV1 cation channel, which also responds to the irritant alkaloid capsaicin and to heat, may be of some interest. We found that TRPV1 (also known as the vanilloid receptor, VR1) is expressed by normal human osteoclasts and that low concentrations of capsaicin strongly activate resorption pit formation in nonacidified conditions (20). It should be noted that ingested capsaicin (e.g., via chili peppers) appears to be almost completely degraded by the stomach and liver and is unlikely to reach the general circulation and bone in significant quantities. The endogenous ligand for TRPV1 may be the endocannabinoid anandamide.
Acidosis and osteoblast function.
Bushinsky and colleagues (21,22) reported that acidosis inhibited osteoblast function by decreasing expression of extracellular matrix genes, including collagen, as well as reducing mineralization. We studied the effects of pH on osteoblast function in our laboratory using bone nodule-forming primary rat osteoblast cultures (23). We found that abundant, matrix-containing mineralized nodules form at pH 7.4 but that acidification progressively reduces mineralization of bone nodules, with complete abolition at pH 6.9 (Fig. 3). Alkaline phosphatase activity, which is required for bone mineralization, peaks strongly near pH 7.4 but is reduced by >90% at pH 6.9, whereas matrix Gla protein, an inhibitor of mineralization, is up-regulated. The same pH reduction is associated with 2- and 4-fold increases in Ca2+ and 
solubility for hydroxyapatite, respectively. However, we also found that osteoblast proliferation and collagen synthesis are unaffected by pH in the range 7.4 to 6.9; moreover, no effect of acidification on collagen ultrastructure and organization is evident. Thus, our results indicate that acidosis exerts a selective, inhibitory action on matrix mineralization that is reciprocal with the osteoclast activation response (23).
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The responses of bone cells to pH changes constitute a homeostatic mechanism that helps to maintain systemic acid-base balance. In acidosis, osteoclast resorptive activity is increased, and the deposition of alkaline mineral in bone by osteoblasts is reduced, to maximize the availability of hydroxyl ions in solution to buffer protons (Fig. 4).
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Our results may help to explain the pathogenesis of a wide range of bone disorders and emphasize the critical role played by the vasculature in bone health. Future therapies for treating bone loss disorders could be based on shifting systemic acid-base balance in the alkaline direction using diet (e.g., via fruit and vegetables and calcium salts) or drugs or by targeting H+-sensing receptors on osteoclasts. Our findings also provide further rationale for the promotion of vascular health via aerobic exercise, avoidance of smoking, and good diet.
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FOOTNOTES |
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2 Financial support: Arthritis Research Campaign, Biotechnology and Biological Sciences Research Council.
3 Author disclosure: T. R. Arnett received a research grant from Novartis Pharmaceuticals.
4 Abbreviations used: 1,25D3, 1,25-dihydroxycholecalciferol; NFATc1, nuclear factor of activated T-cells, cytoplasmic 1; OGR1, ovarian cancer G protein-coupled receptor; RANKL, receptor activator of nuclear factor B ligand; TDAG8, T cell death-associated gene 8; TRPV1, transient receptor potential vanilloid.
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LITERATURE CITED |
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), whereas at lower pH, bone mineral is mobilized (i.e., dissolved) to release OH– ions (
) into the extracellular fluid and circulation to correct systemic acidosis. [Adapted from Arnett (