Nitric oxide regulates alkaline phosphatase activity in rat fracture callus explant cultures

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Nitric Oxide Regulates Alkaline Phosphatase Activity in Rat Fracture Callus Explant

Cultures

Heeja Namkung-Matthai l ,2, Ashish Diwan2, Rebecca S Mason3, George MurreU2,

Terry Diamond I,

IDepartment of Endocrinology and 20rthopaedic Research Institute, St. George

Hospital, University of New South Wales, Kogarah, NSW 2217, Australia,

3Department of Physiology and Institute for Biomedical Research, University of

Sydney, NSW 2006, Australia

IAddress correspondence to: Dr. T. Diamond, Department of Endocrinology, St. George Hospital,

University of New South Wales, Kogarah, NSW 2217, Australia; Phoue: +61-2-93502772; Fax: +61­

2-9350 3966; E-mail: terryd@hare.nel.au

Running Title: Nitric oxide on alkaline phosphatase activity during fracture healing

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ABSTRACT

Nitric oxide (NO) is synthesised by a group of enzymes called nitric oxide

synthases (NOS) and oxidizes to its stable endproducts nitrite (N02-) and nitrate (N03­

). We have previously reported in an in vivo rat model that NO is an important regulator

for rat bone fracture healing (I). This study examines the effects of NO on alkaline

phosphatase (ALP) activity in a rat fracture callus explant culture system. Explants of

rat femoral fracture callus from days 4, 7, 14 and 28 post fracture induced N02- release

and ALP activity in a biphasic temporal manner, with the highest activity on day 7 and

the lowest activity on day 14. Inhibition ofNOS by co-incubation with a NOS inhibitor

S-(2-Aminoethyl) isothiouronium bromide hydrobromide (AETU) inhibited ALP

activity by an average 50% at each time point (p < 0.01). Supplementation with NO

donor 3-morpholinosydnonomine hydrochloride (SIN-I) at low doses (25 and 0.025

IlM) increased ALP activity by 20% (p < 0.01). ALP mRNA and histochemical ALP

activity was localised to osteoblast-like and chondrocyte-like cells within fracture callus.

The current study provides evidence that NO plays a regulatory role in ALP

activity during rat fracture healing.

Key words: fracture healing, nitric oxide, alkaline phosphatase activity, bone

regeneration, callus

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