Clonidine increases bone resorption in humans

Twelve healthy female and male volunteers aged 18–70 years were recruited for this crossover study. Exclusion criteria for participation were (1) contra-indications to clonidine, i.e., hypersensitivity, pulse rate <50 per minute, and use of antihypertensive drugs or drugs with negative chronotropic effects on the heart; (2) any medication or disease influencing bone turnover; (3) blood pressure <110/70 mmHg; and (4) pregnancy. At baseline, we took a complete history and measured body weight, height, and blood pressure. A venous blood sample was drawn after an overnight fast to determine serum concentrations of calcium, albumin, phosphate, parathyroid hormone (PTH), 25-hydroxyvitamin D (25(OH)D), creatinine, and alkaline phosphatase.

This open-label crossover trial was performed at the Department of Endocrinology and Metabolism of the Academic Medical Center of the University of Amsterdam (AMC/UvA) in the Netherlands. All participating subjects were studied two times (clonidine and control) and served as their own control. The 2 study days were separated by a washout period of at least 1 week. Participants were randomly allocated to clonidine followed by no intervention or no intervention followed by clonidine using a computer-generated randomization list (nQuery Advisor version 7.0, Statistical Solutions, Cork, Ireland). An antecubital vein of the arm was cannulated for all blood sampling. A baseline blood sample was drawn between 8:30 and 9:00 h to determine bone turnover markers (C-terminal cross-linking telopeptides of collagen type I (CTx) and procollagen type 1 N propeptide (P1NP)), normetanephrine, and creatinine concentrations in plasma or serum, after which participants received either a single oral dose of clonidine (a selective alpha-2-adrenoceptor agonist) 0.3 mg or no intervention at 9:00 h. Five blood samples were drawn during the following 6 h to determine bone turnover markers, normetanephrine, and creatinine concentrations. All blood samples were centrifuged within 30 min and stored at −20 ° C until analysis. Participants were fasted throughout the experiment but were allowed to drink water. They were restricted to bed rest with minimal physical activity. During the experiment, blood pressure and heart rate were monitored every 15 min, with a Microlife® BP 3 AC1-1 automatic device. The study was approved by the Institutional Review Board of the Academic Medical Center (AMC) in Amsterdam and was carried out in compliance with the Helsinki Declaration. All subjects agreed to participate in the study and written informed consent was obtained from all participants. The trial was registered in the Netherlands Trial Register (NTR3762) before start of the study.

Serum C-terminal cross-linking telopeptides of collagen type I (CTx), a marker of bone resorption, and serum procollagen type 1 N propeptide (P1NP), a marker of bone formation, were measured as bone turnover markers. CTx and P1NP concentrations were measured using an immunoassay (Modular Analytics E 170; Roche Diagnostics Corporation, Indianapolis, IN; and Orion Diagnostica, Espoo, Finland, respectively), as described earlier [27]. Plasma normetanephrine was determined by automated online solid phase extraction HPLC-tandem mass spectrometry as described by De Jong et al. [28]. Serum creatinine concentrations were measured on a Hitachi Modular 8000 (C602 and C702) (Roche Diagnostics, Mannheim, Germany). Assays were performed in duplicate and samples from a single subject were assayed within the same analytical batch. Inter-assay coefficients of variation were as follows: CTx 3 %, P1NP 8 %, and normetanephrine 7.7 %.

CD14⁺ monocytes were isolated from human buffy coats (Sanquin, Amsterdam, Netherlands) obtained from healthy volunteers who provided written informed consent. Briefly, human buffy coats were diluted with phosphate-buffered saline (PBS) containing 1 % citrate (1:1) and then spun down (800 g, 30 min, without brake) in Ficoll gradient solution. The interphase containing the peripheral blood mononuclear cells was collected and washed four times in 0.5 % bovine serum albumin buffer (2 mM EDTA, phosphate-buffered saline) and then passed through a cell strainer (40 mm) to ensure the recovery of a clean mononuclear cell population. Cells were counted using a Muse cell counter (Merck Millipore, Darmstadt, Germany) and incubated with CD14 antibody tagged microbeads (Miltenyi Biotec) (1 × 10⁷ cells in 20 μL microbead solution) for 15 min at 4 °C and then sorted using a manual MACS magnetic cell sorter (Miltenyi Biotec) according to the manufacturer’s instructions. This method is frequently used to isolate osteoclast precursors [29]. The purity of the cells was confirmed to be 86 % by flow cytometry (Accuri C6, Becton Dickinson) using a FITC-labeled CD14 antibody and mouse IgG2a isotype antibody (both Miltenyi Biotec) as control. Purified CD14⁺ cells were suspended in culture medium consisting of αMEM (Life Technologies) supplemented with 10 % fetal calf serum (HyClone I), 1 % penicillin/streptomycin (Sigma-Aldrich), and 25 ng/mL macrophage colony-stimulating factor (M-CSF) (R&D systems). Bovine cortical bone slices of 650-μm thickness were soaked in a 96-well plate with 100 μL culture medium for 2 h prior to seeding. Next, the medium was removed and 75 μL of medium containing 25 ng/mL M-CSF was added to the bone slices, upon which 75 μL of cell suspension containing 1.5 * 10⁵ cells was added. After 3 days of culturing with M-CSF, the medium composition was changed to 10 ng/mL M-CSF and 2 ng/mL receptor activator of nuclear factor kappa-B ligand (RANKL) (R&D Systems). All cultures were maintained at 37 °C, in a humidified atmosphere under 5 % CO₂. Culture media were replaced every 3–4 days.

A time titration experiment was performed first, to determine the appropriate point in time to add clonidine. In this experiment, CD14⁺ cells were cultured for 28 days, with collection of samples for tartrate-resistant acid phosphatase (TRAcP) staining and bone resorption at days 7, 10, 14, 17, 21, 24, and 28. On day 17, wells were dissolved in RNA lysis buffer from the RNeasy Mini Kit (Qiagen, Hilden, Germany) and stored at −80 °C for RNA isolation to determine messenger RNA (mRNA) expression of osteoclast-related genes (tartrate-resistant acid phosphatase (TRAP), cathepsin K (CTSK), calcitonin receptor (CALCR), and dendritic-cell-specific transmembrane protein (DCSTAMP)) and of alpha-2-adrenergic receptor subtypes (ADRA2A, ADRA2B, ADRA2C). Based on the bone resorption results from this experiment (data not shown), we decided to add clonidine after 14 days of culturing.

In a separate experiment, clonidine (Sigma-Aldrich) was added to the culture medium after 14 days of culturing in increasing concentrations (10⁻⁹, 10⁻⁷, and 10⁻⁵ M). These concentrations were chosen based on peak plasma concentrations achieved after a single oral dose of clonidine 0.3 mg [30, 31] and concentrations used in a previous in vitro study using mouse osteoclasts [9]. Wells containing the solvent (water) used for clonidine served as controls. After 14, 21, and 28 days of culturing, wells were washed with PBS and the cells were either fixed in 4 % formaldehyde (used for TRAcP staining) or washed with water (used for bone resorption) and stored at 4 °C.

All cultures were run at least in fivefold (for TRAcP staining), in tenfold (for bone resorption), and in sixfold (for RNA analysis).

Osteoclast-like cells were defined as multinucleated (three or more nuclei) tartrate-resistant acid phosphatase (TRAcP)–positive cells. After 14, 21, and 28 days of culturing, TRAcP positivity was determined by enzyme histochemistry (Leukocyte Acid Phosphatase Staining Kit, Sigma-Aldrich). Nuclei were stained with diamidino-2-phenylindole dihydrochloride (DAPI). Per bone slice, the number of TRAcP-positive cells with three or more nuclei were counted at a ×200 magnification. We considered multinucleated cells with three to five nuclei as small osteoclasts and multinucleated cells with more than five nuclei as large osteoclasts. The data were expressed as number of TRAcP⁺ multinucleated cells (MNCs) per square centimeter.

In order to evaluate osteoclast activity, resorption was analyzed after a culture period of 14, 21, and 28 days. Cells were lysed with demineralized water, after which bone slices were sonicated for 30 min in 10 % ammonium to remove the cell remnants. Bone slices were then thoroughly washed with demineralized water and subsequently incubated in a 10 % saturated alum (KAl(SO₄)₂·12H₂O) solution for 10 min. Finally, the bone slices were washed again with demineralized water and the resorption pits were stained with Coomassie brilliant blue. An entire bone slice was micrographed at a ×200 magnification, using a digital camera, mounted on a light microscope (both from Leica, Wetzlar, Germany). We reconstructed the bone slices by merging the micrographs using Adobe Creative Suite Bridge (San Jose, CA, USA). The resorbed area was measured using Image Pro-Plus Software (Media Cybernetics, Silver Spring, MD, USA) and expressed as percentage of the total surface area.

Gene expression of osteoclast-related genes (TRAP, CTSK, CALCR, and DCSTAMP) and of alpha-2-adrenergic receptor subtypes (ADRA2A, ADRA2B, ADRA2C) was quantified by PCR at 17 days of culturing. Total RNA was extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. Reverse transcription of RNA was performed using the MBI Fermentas cDNA synthesis kit (Fermentas, Lithuania), using both the Oligo(dT)18 and the D(N)6 primers. Quantitative PCR (qPCR) was performed on the ABI PRISM 7000 Sequence Detection System as described previously [32]. Primers were designed using the Primer Express software (version 2.0, Applied Biosystems, Foster City, CA, USA) (Table 1). To avoid amplification of genomic DNA, primers were designed such that each amplicon spanned at least one intron. The PCR reactions of the different amplicons had equal efficiencies as determined by serial dilutions of a calibrator sample. For the PCR analysis, porphobilinogen deaminase (PBGD) was used as housekeeping gene. Expression of this gene was not affected by the experimental conditions. Samples were normalized for the expression of PBGD by calculating the ΔCt (Ct_{gene of interest} − Ct_PBGD). Expression of the different genes is expressed as 2^−(ΔCt).