α₁ adrenergic receptor agonist, phenylephrine, actively contracts early rat rib fracture callus ex vivo

Abstract

Early, soft fracture callus that links fracture ends together is smooth muscle-like in nature. We aimed to determine if early fracture callus could be induced to contract and relax ex vivo by similar pathways to smooth muscle, that is, contraction via a 1 adrenergic receptor (a 1 AR) activation with phenylephrine (PE) and relaxation via b 2 adrenergic receptor (b 2 AR) stimulation with terbutaline. A sensitive force transducer quantified 7 day rat rib fracture callus responses in modified Krebs-Henseliet (KH) solutions. Unfractured ribs along with 7, 14, and 21 day fracture calluses were analyzed for both a 1 AR and b 2 AR gene expression using qPCR, whilst 7 day fracture callus was examined via immunohistochemistry for both a 1 AR and b 2 AR-immunoreactivity. In 7 day callus, PE (10 À6 M) significantly induced an increase in force that was greater than passive force generated in calcium-free KH (n ¼ 8, mean 51% increase, 95% CI: 26-76%). PE-induced contractions in calluses were attenuated by the a 1 AR antagonist, prazosin (10 À6 M; n ¼ 7, mean 5% increase, 95% CI: 2-11%). Terbutaline did not relax callus. Gene expression of a 1 ARs was constant throughout fracture healing; however, b 2 AR expression was down-regulated at 7 days compared to unfractured rib ( p < 0.01). Furthermore, osteoprogenitor cells of early fibrous callus displayed considerable a 1 AR-like immunoreactivity but not b 2 AR-like immunoreactivity. Here, we demonstrate for the first time that early fracture callus can be pharmacologically induced to contract. We propose that increased concentrations of a 1 AR agonists such as noradrenaline may tonically contract callus in vivo to promote osteogenesis. ß

Noradrenaline stimulates cell proliferation by suppressing potassium channels via Gi/o‐protein‐coupled α1B‐adrenoceptors in human osteoblasts

“…There is also a report that a1-adrenoceptor blockade increased the risk of hip/ femur fracture (Souverein et al, 2003). Phenylephrine, an a1-adrenoceptor agonist, promotes osteogenesis in bone fracture healing (McDonald et al, 2011). All these reports suggest the possibility that activation of a1-adrenoceptors could lead to a treatment for osteoporosis.…”

Loss of Gi G-Protein-Coupled Receptor Signaling in Osteoblasts Accelerates Bone Fracture Healing

“…(27,35) Although these prior results indicated that PTH could activate the bone repair process, the fact that PTHR1 and other GPCRs can each activate G s , G i , and G q GPCR signaling pathways (36) poses a challenge to identifying how the G s or G i pathways specifically regulate fracture repair. In addition, the balance of G s and G i signaling may contribute to other observed effects of other GPCRs on bone formation and fracture healing, such as the promotion of osteogenesis by predominantly G s -coupled b-adrenergic receptors (37,38) and prostaglandin E2 receptors. (39) Histomorphometry of nonstabilized fracture callus shows total callus volume was not significantly different between the control and Col1/Rs1-late mice except at day 7.…”

Transient expression of myofibroblast-like cells in rat rib fracture callus

“…RNA was extracted from samples as described previously (McDonald et al 2011). Reverse transcription was performed using the SuperScript First-Strand Synthesis System according to the manufacturer's instructions (Invitrogen, Mulgrave, Victoria, Australia).…”

“…The contractile microfilament, alpha smooth muscle actin (αSMA), is abundantly expressed in early fibrous callus and is recognized as a marker of osteoprogenitor cell populations (Kinner et al 2002, Dooley et al 2004, Kalajzic et al 2008). Furthermore, recent findings from our laboratory suggest that this αSMA expression translates into functional smooth muscle-like passive viscoelastic and active contractile properties of early rat rib fracture callus (McDonald et al 2009, 2011)…”

“…We have previously speculated that the cells responsible for such contractile characteristics are myofibroblast-like (McDonald et al 2011). Myofibroblasts are phenotypically intermediate between smooth muscle cells and fibroblasts, and have a well-documented role in facilitating wound contraction in healing of soft-tissue wounds (Gabbiani 2003, Desmouliere et al 2005).…”