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The osteoclast is unique in its ability to resorb bone, and excessive osteoclastic activity has been implicated in osteoporosis, Paget disease of bone, rheumatoid arthritis, and the growth of metastases in bone. The activity of this cell is controlled by the main circulating inhibitor, calcitonin, in association with locally produced modulators. We show that nitric oxide (NO) may be an important member of the latter group. NO is produced by the vascular endothelium and nervous system and is involved in both neurotransmission and the regulation of blood pressure. However, our results show that the autocoid is also a potent inhibitor of osteoclast func- (7), the main circulating regulator of osteoclast function (9). We have also shown that the concentration of calcium ions, generated as a result of osteoclast activity, can directly regulate osteoclast function (10), providing a possible feedback control. However, locally produced cytokines are also important in the control of osteoclast activity (11), although less progress has been made in identifying the main local regulators or in understanding their mode of action. Because of the abundance of endothelial cells in bone marrow and their proximity to bone cells (12), we have applied our techniques to an examination of the effects of nitric oxide (NO). This autacoid appears to play an important role in intercellular communication in neutrophils, brain tissue, renal epithelial cells, mast cells, and autonomic nerves, in addition to fulfilling its known function as a vasodilator produced by the vascular endothelium (13). We show that NO also exerts a powerful inhibitory effect on the bone-resorbing activity of the osteoclast, suggesting that the marrow endothelial cell may have a physiological role in modulating osteoclast activity. MATERIALS AND METHODSChemicals. Human calcitonin and the 1,7-aminosuberic acid analogue of eel calcitonin (eCT) were gifts from CIBAGeigy and ISF (Milan, Italy), respectively. The peptides were dissolved in distilled deionized water containing acetic acid (0.001%, vol/vol; Aristar grade; BDH) and bovine serum albumin (0.01%, wt/vol; Sigma; recrystallized), then lyophilized, and stored at -70°C. Indo-1 AM was purchased from Molecular Probes. 3-Morpholinosydnonimine hydrochloride (SIN-1) was a gift from Casella AG, Pharmaforschung Galenik (Frankfurt).A saturated solution of NO gas (3 mM) was prepared in a Wheaton flask in 25 ml of distilled water at 0°C after deoxygenation by bubbling with helium for 30 min. Appropriate volumes (3 and 10 ,ul) offreshly prepared solution were added to 1 ml ofcell culture medium (see below) immediately before use to produce final concentrations of 9 and 30 ,uM.Tissue Culture Materials. Tissue culture media and heatinactivated fetal calf serum were purchased from Flow Laboratories. Osteoclasts were isolated in medium 199 buffered with Hepes and supplemented with fetal calf serum (10%o vol/vol), benzyl penicillin (Glaxo; 100 units/liter), and streptomycin (Glaxo; 100 mg/liter) (referred to as medium...

Nitric oxide (NO) produces rapid osteoclast detachment and contraction in vitro, and this effect is accompanied by a profound inhibition of bone resorption. Work by others has confirmed these findings in vivo: inhibition of NO synthase [NOS; L-arginine, NADPH: oxygen oxidoreductase (NO-forming), EC 1.14.13.39] in normal rats is followed by increased bone resorption reflected by a marked loss in bone mineral density. In our present study, immunocytochemistry and Northern blotting show the presence of the constitutive calcium-sensitive NOS isoform (cNOS) in normal rat osteoclasts and in the human preosteoclast cell line (FLG 29.1 by normal rat osteoclasts treated with LPS or IFN-'y. In contrast, the nonselective NOS inhibitor NG-monomethyl-Larginine inhibits resorption by untreated neonatal rat osteoclasts. Thus, osteoclast function may require intermittent calcium-stimulated increases in NO production by cNOS against a basal inhibitory background activity of the iNOS isoform. However, bone resorption depends on precursor replication and on the activity of the mature cells, and we found that the NO donor 3-morpholinosydnonimine (SIN-1) (50 ,uM) profoundly depressed replication in the human preosteoclast line. Taken together, these results strongly suggest that NO maintains a central control of bone resorption in both avian and mammalian species by exerting a powerful tonic restraint of osteoclast numbers and activity. The presence of NOS in human cells implies a similar function in man and that conventional views of calcium homoeostasis and skeletal metabolism will need substantial revision. Since NO also influences behavior of the osteoblast, the boneforming cell, in vitro, a similar effect in vivo might imply a general influence on bone remodeling.Nitric oxide, or NO, secreted by the endothelium exerts an overriding control of blood pressure and blood flow by producing a dominant tonic dilatation of arterial muscle. To this crucial physiological role is added a number of other important functions. In the central nervous system, NO potentiates long-term memory; in the periphery it is the transmitter in the nonadrenergic noncholinergic (NANC) inhibitory nerves of the gastrointestinal and urogenital tracts. In contrast, the macrophage uses NO to kill invading microorganisms. NO is produced by several NO-synthase [NOS; L-arginine, NADPH: oxygen oxidoreductase (NO-forming), EC. 1.14.13.39] isoforms that require the same cofactors but vary in their response to calcium, mode of regulation, and tissue specificity (1). Constitutive isoforms of NOS have been identified in brain (2) and endothelium (3), while in macrophages (4) and hepatocytes (5) NOS transcripts of inducible NOS (iNOS) isoforms are detected only after treatment with cytokines or lipopolysaccharide (LPS). However, this gas radical now appears to be involved in still another function-that of control of osteoclastic activity. The osteoclast is the only cell in the body that can resorb bone and, together with the kidney, this cell plays the major role...

Nitric oxide (NO) has been implicated in the local regulation of bone metabolism. However, the contribution made by specific NO synthase (NOS) enzymes is unclear. Here we show that endothelial NOS gene knockout mice (eNOS؊/؊) have marked abnormalities in bone formation. Histomorphometric analysis of eNOS؊/؊ femurs showed bone volume and bone formation rate was reduced by up to 45% (P < 0.01) and 52% (P < 0.01), respectively. These abnormalities were prevalent in young (6 to 9 weeks old) adults but by 12 to 18 weeks bone phenotype was restored toward wild-type. Dual energy X-ray absorptiometry analysis confirmed the age-related bone abnormalities revealing significant reductions in femoral (P < 0.05) and spinal bone mineral densities (P < 0.01) at 8 weeks that were normalized at 12 weeks. Reduction in bone formation and volume was not related to increased osteoclast numbers or activity but rather to dysfunctional osteoblasts. Osteoblast numbers and mineralizing activity were reduced in eNOS؊/؊ mice. In vitro, osteoblasts from calvarial explants showed retarded proliferation and differentiation (alkaline phosphatase activity and mineral deposition) that could be restored by exogenous administration of a NO donor. These cells were also unresponsive to 17␤-estradiol and had an attenuated chemotactic response to transforming growth factor-␤. Bone is a vital dynamic connective tissue that has evolved to maintain a balance between its two major functions: provision of mechanical integrity for locomotion and modulation and control of mineral homeostasis. 1 Mineralized bone is continuously resorbed by osteoclasts and new bone is formed by osteoblasts. This process, known as bone remodeling, is highly regulated with maintenance of normal integrity and structure. 2 Systemic hormones including calcitonin, parathyroid hormone, and sex steroids, particularly estrogen, are known to be important regulators of bone cell function. Their effects on bone turnover are in general exerted by activation of local mediators and second messengers present within bone cells. 3 Recent investigations have focused on the role of nitric oxide (NO) as one of these possible local regulators of bone metabolism and bone cell activity. NO is a shortlived radical gas generated from L-arginine by nitric oxide synthase (NOS) isoenzymes. 4 Three distinct isoforms of NOS have been identified: a neuronal form (type I; nNOS) originally isolated from brain, 5 an endothelial form (type III; eNOS) originally isolated from bovine aortic endothelial cells, 6 and an inducible form (type II; iNOS) originally isolated from murine macrophages. 7 Both eNOS and nNOS are expressed constitutively and are characterized by highly regulated rapid but low-output NO production. 4 In contrast the iNOS pathway is generally only activated after stimulation by certain pro-inflammatory cytokines such as interferon-␥, interleukin-1␤, and tumor necrosis factor-␣. The inducible NOS isoform is characterized by production of persistent and high concentrations of NO. 8 There is now am...

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The rat calcitonin gene has recently been shown to encode a novel peptide (rat calcitonin gene-related peptide, rCGRP) thought to be produced in nervous tissue after tissue-specific RNA processing. This peptide has so far been identified only in rat tissue, by immunocytochemistry and immunoassay. We now report the isolation of a related (89% homology) peptide from human tissue (hCGRP) which we have sequenced using a novel mass spectrometric approach, fast atom bombardment (FAB) mapping. The human peptide differs significantly from the predicted rCGRP structure in four positions in the amino acid sequence (three effecting charge changes), and the presence of a disulphide bridge and an amide, surmised in the rat work, is proven in the hCGRP molecule. hCGRP was present in plasma from 10 patients with medullary thyroid carcinoma (MTC) and in 6 MTC tumours removed at surgery, suggesting the tissue distribution may differ from that in the rat where the peptide is reported to be absent from thyroid tissue. hCGRP is shown to have biological activity and it is possible that its presence in MTC plasma may be responsible for some of the symptoms in this disease.

Abstract-Proteinuria is associated with adverse cardiovascular and renal outcomes that are not prevented by current treatments.Endothelin 1 promotes the development and progression of chronic kidney disease and associated cardiovascular disease. We, therefore, studied the effects of selective endothelin-A receptor antagonism in proteinuric chronic kidney disease patients, assessing proteinuria, blood pressure (BP), and arterial stiffness, key independent, surrogate markers of chronic kidney disease progression and cardiovascular disease risk. In a randomized, double-blind, 3-way crossover study, 27 subjects on recommended renoprotective treatment received 6 weeks of placebo, 100 mg once daily of sitaxsentan, and 30 mg once daily of nifedipine long acting. Twenty-four-hour proteinuria, protein:creatinine ratio, 24-hour ambulatory BP, and pulse wave velocity (as a measure of arterial stiffness) were measured at baseline and week 6 of each treatment. In 13 subjects, renal blood flow and glomerular filtration rate were assessed at baseline and week 6 of each period.