Simultaneous measurement of bone resorption and collagen synthesis in neonatal mouse calvaria☆
Abstract
A tissue culture technique which permits the simultaneous measurement of collagen synthesis and bone resportion has been developed. Cultured neonatal mouse calvaria undergo resorption when stimulated by a number of agents including parathyroid hormone, vitamin D, and prostaglandin E2. Mouse calvaria are of sufficient size to measure the extent of proline incorporation into collagenase-digestible protein. Four chemically diverse stimulators of bone resorption were tested for their effect on collagen synthesis. For each stimulator tested, the dose-response relationships for the stimulation of resorption and the inhibition of collagen synthesis were found to coincide.
References (19)
- P.A. Price et al.
J. Biol. Chem
(1980) - D.W. Rowe et al.
J. Biol. Chem
(1982) - T.J. Hefley et al.
Exp. Cell Res
(1983) - B. Peterkofsky
- W.H. Porter et al.
J. Biol. Chem
(1971) - L.G. Raisz et al.
Prostaglandins
(1974) - Y.S. Chyun et al.
Prostaglandins
(1984) - G.A. Rodan et al.
Calcif. Tissue Int
(1981) - T.J. Chambers et al.
Clin. Orthop. Relat. Res
(1980)
Cited by (20)
Osteo-Odyssey: A Memoir
2013, Osteoporosis: Fourth EditionAlmost 50 years ago, in the fall of 1963, I entered the bone field when I became Larry Raisz's first Ph.D. postdoctoral fellow. It was not because I had been interested previously in osteoporosis or even in bone. In college, I had particularly enjoyed invertebrate zoology. Not a good predictor of a career in bone research. My Ph.D. in pharmacology from the University of Michigan was on carbon tetrachloride hepatotoxicity, examining effects on oxidative phosphorylation and triglyceride metabolism. Again not very bone related. But I did have a continuing thread of interest in endocrinology, writing my M.S. qualifying exam on thyroid hormone analogs, taking an experimental endocrinology elective as a graduate student, being intrigued by the permissive effect of adrenocortical steroids on the fatty liver elicited by carbon tetrachloride, and going to Woods Hole (MA, US) for the comparative endocrinology course. I decided to seek postdoctoral training in endocrine pharmacology, which gave me enough latitude, as one-half of a dual career couple, to explore multiple possibilities. One of my postdoc interviews was at a Federation of American Societies for Experimental Biology (FASEB) meeting in Atlantic City, where the representative of the pharmacology department at the University of Rochester was Larry Raisz, a physician who had recently joined their department to establish a clinical pharmacology program. I was intrigued by Larry's enthusiasm, and decided it would be interesting to learn more about parathyroid hormone (PTH) and bone. I was in Larry's laboratory for less than 1 year, again due to dual-career-couple factors, but the months there ensnared me into a rapidly expanding field that presented many interesting questions. It also provided me with the opportunity to interact with the leaders of the field as they became ascendant, and with bright and dedicated young investigators from the many areas that converged on bone. Ultimately, it led me to become involved in the organizations upon which science is dependent, including professional organizations, federal agencies, and foundations involving interactions between medical professionals and lay groups.
Intracellular measurement of prostaglandin E<inf>2</inf>: Effect of anti- inflammatory drugs on cyclooxygenase activity and prostanoid expression
1999, Analytical BiochemistryCyclooxygenase (COX) converts arachidonic acid to prostaglandin (PG) H2, which is further metabolized to various prostaglandins, prostacyclin and thromboxane A2. COX exists in at least two different isoforms. COX-1 is constitutively expressed, whereas COX-2 is induced by proinflammatory stimuli. Prostaglandin E2 is a major metabolite of COX activation. In order to compare the activity of target ligands and COX inhibitors on PGE2 synthesis and release, the responsiveness of several cell lines to the calcium ionophore A23187, bacterial lipopolysaccharide (LPS), nonsteroidal anti-inflammatory drugs (NSAIDs), and the glucocorticoid, dexamethasone, were investigated. For intracellular measurements, the culture supernatant was aspirated, and the cells were thoroughly washed and lysed with dodecyltrimethylammonium bromide. Intracellular and secreted PGE2 were measured with an enzyme immunoassay. A23187 and LPS increased intracellular PGE2 in a dose-dependent manner. Kinetic experiments with A23187-stimulated mouse 3T3 fibroblast cells revealed a distinct biphasic response in COX activity. In the presence of NSAIDs or dexamethasone, there was a dose-dependent inhibition in intracellular PGE2 with A23187-stimulated 3T3 cells. Inhibitory studies demonstrated an apparent increased sensitivity of COX activity to the action of inhibitors when measuring intracellular PGE2 compared with using cell culture supernatants. Indeed, intracellular PGE2 levels were comprehensively reduced in the presence of low concentrations of inhibitor. The utilization of cell culture lysates and, in particular, measurement of intracellular PGE2 should prove useful for identifying new COX inhibitors.
Induction of Cyclo-Oxygenase and Nitric Oxide Synthase in Inflammation
1996, Advances in PharmacologyThis chapter focuses on the role of prostanoids and NO at each stage of the inflammatory response with particular emphasis given to the cellular source and the factors that may modulate the activity of their respective enzymes. Inhibition of prostanoid formation by use of nonsteroidal anti-inflammatory drugs (NSAIDs) ameliorates the classical signs of inflammation, indicating their pivotal role in the inflammatory response. Recently, a second isoform of cyclooxygenase (COX), the enzyme that liberates the prostanoids, has been identified. This discovery has given new impetus as to the role of COX isoforms in inflammation and has fueled the search for selective inhibitors free from side effects. Once liberated, arachidonic acid is converted to the biologically active PGs and thromboxanes (TXs), collectively termed prostanoids, by the enzyme COX, also known as prostaglandin H synthase or prostaglandin endoperoxidase synthase. COX has two functions: first, cyclo-oxygenase activity that catalyzes PGG2 formation, and second, peroxidase activity that reduces the 15-hydroperoxyl group of PGG, to PGH2. The chapter keeps space for discussion on both COX-1 and COX-2. The biological activity of the elusive molecule, termed endothelium-derived relaxing factor (EDRF), involved in vascular relaxation, could be accounted for by nitric oxide (NO). It was found that NO, synthesized by endothelial cells (ECs), acted as an intercellular effector molecule, causing vasodilatation by activation of guanylate cyclase and the elevation of cGMP levels in vascular smooth muscle cells. Although NO is indubitably involved in inflammation its role cannot be defined as pro- or anti-inflammatory but rather depends on the prevailing circumstances. The products of the NOS and COX pathways as well as having potent effects on various cellular systems can also modulate the activity of their respective enzymes. Although the mutual effects between COX activity and action of NO effects are not clear cut as both stimulatory and inhibitory actions have been ascribed.
Chapter 21. Recent Advances in Bone Metabolism and Osteoporosis Research
1991, Annual Reports in Medicinal ChemistryAlthough the term “osteoporosis” (OP) is frequently used in reference to the process of abnormally accelerated bone loss (osteopenia), particularly that associated with the onset of menopause, it is, in fact, a heterogeneous disorder clinically defined as a predisposition to bone fracture. It is the pathological end result of multiple causes and risk factors that include peak bone mass prior to the onset of aging-related bone loss and, in women, its exacerbation by the loss of ovarian hormonal support. There has been an exponential growth in the fundamental knowledge of bone metabolism and how defects in this system are reflected in disorders, such as OP, Paget's disease, and osteomalacia. This chapter discusses some of the new developments in bone research at both the basic and clinical levels. The chapter explains bone metabolism with mechanistic insights that involves growth factors, cytokines, noncollagenous bone protein, bone cell estrogen receptor, proton pump, calcitonin, and superoxide. Clinical developments include current therapies and recent therapies on osteoporosis diagnosis and treatment, hormone replacement, bisphosphonates (BP), calcitonin, and thiazide therapies. Advancements in clinical and preclinical research have resulted in tests for the identification of antiresorptive agents, bone formation inducing agents, bisphosphonate modes of action, enzyme inhibitors, prostaglandins, and other agents.
We have developed a method that allows us to measure bone resorption and formation simultaneously in the parietal bones from 22-day fetal rat calvaria. Parietal bones labeled with 45Ca, by injection of the mother, were cultured for 72 h with parathyroid hormone (PTH, bovine 1–34, 1.56 nM) or prostaglandin E2 (PGE2, 100 nM), in the presence or absence of indomethacin (Indo, 1 μM) or corticosterone (Cort, 1 μM). Two hours prior to the end of the culture, the bones were pulsed with [3H]-proline or [3H]-thymidine. Resorption was assessed as the percent of 45Ca released into the medium. Incorporation of [3H]-proline into collagenase digestible protein (CDP) and of [3H]-thymidine into DNA (TDR) were measured to assess collagen and DNA synthesis, respectively. Basal % 45Ca release was 16 ± 1% and was significantly decreased by Indo and Cort. Cort decreased TDR and CDP while Indo did not. PTH and PGE2 significantly increased %45Ca release, and this was not blocked by Indo. However, in the presence of Cort, only PTH increased %45Ca release while PGE2 did not. PGE2 increased TDR under all culture conditions while PTH increased TDR only in the presence of Cort. While PTH and PGE2 had the same effects on bone resorption, they had different effects on CDP. PGE2 increased CDP in the presence of Indo or Cort but PTH did not. Thus, this model allows us to study bone resorption, collagen synthesis, and DNA synthesis simultaneously. We have also shown that PTH and PGE2 differ in their sensitivity to inhibition of resorption by Cort and in their effects on bone formation.
Bone repletion in vitro: Evidence for a locally regulated bone repair response to PTH treatment
1988, BoneNormally bone formation and resorption are balanced by coupling, but in some conditions such as dietary Ca deficiency, bone resorption exceeds formation, resulting in bone loss (termed bone depletion in previous animal studies). When conditions causing depletion return to normal, a compensatory decrease in resorption and increase in formation occurs, leading to replacement of the lost bone. To test if this recovery process, termed bone repletion, might be locally regulatedupling we determined whether cellular and metabolic changes associated with repletion in vivo would as in co occur in vitro in neonatal mouse calvaria.
To increase resorption and decrease formation, serumfree cultures were treated with parathyroid hormone (10 nM bovine PTH1–84). Although formation was inhibited ([3H]proline incorporation into [3H]hydroxyproline), the number of bone cells increased during PTH treatment. To simulate repletion, PTH was removed after 3–9 days. Within 6 days of removal of PTH, resorption (osteoclast numbers and 45Ca release) decreased to control levels and bone formation increased to exceed untreated control levels. Autoradiographs of [3H]proline incorporation suggested an increase in the number of active bone forming cells (compared to untreated controls) after removal of PTH. These cellular and metabolic changes were similar to changes which occur during depletion and repletion in vivo. The results support the hypothesis that reversal of the resorptive processes initiated by PTH in organ cultures can occur in the absence of circulating factors. The apparent increase in the components of bone formation that were observed after PTH withdrawal may have resulted from generation of increased numbers of osteoblastic cells during PTH treatment.
- ☆
This work was supported by NIH Research Grants AM31246 and AM11262.