Suppression of mammalian bone growth by membrane transport inhibitors.
Bone lengthening during skeletal growth is driven primarily by the controlled enlargement of growth plate (GP) chondrocytes. The cellular mechanisms are unclear but membrane transporters are probably involved. We investigated the role of the Na(+)/H(+) antiporter (NHE1) and anion exchanger (AE2) in bone lengthening and GP chondrocyte hypertrophy in Sprague-Dawley 7-day-old rat (P7) bone rudiments using the inhibitors EIPA (5-(N-ethyl-N-isopropyl)amiloride) and DIDS (4,4-diidothiocyano-2,2-stilbenedisulphonate), respectively. We have also determined cell-associated levels of these transporters along the GP using fluorescent immunohistochemistry (FIHC). Culture of bones with EIPA or DIDS inhibited rudiment growth (50% at approx. 250 and 25 µM, respectively). Both decreased the size of the hypertrophic zone (P < 0.05) but had no effect on overall length or cell density of the GP. In situ chondrocyte volume in proliferative and hypertrophic zones was decreased (P < 0.01) with EIPA but not DIDS. FIHC labeling of NHE1 was relatively high and constant along the GP but declined steeply in the late hypertrophic zone. In contrast, AE2 labeling was relatively low in proliferative zone cells but increased (P < 0.05) reaching a maximum in the early hypertrophic zone, before falling rapidly in the late hypertrophic zone suggesting AE2 might regulate the transition phase of chondrocytes between proliferative and hypertrophic zones. The inhibition of bone growth by EIPA may be due to a reduction to chondrocyte volume set-point. However the effect of DIDS was unclear but could result from inhibition of AE2 and blocking of the transition phase. These results demonstrate that NHE1 and AE2 are important regulators of bone growth.
Centre for Integrative Physiology, School of Biomedical Sciences, George Square, University of Edinburgh, Edinburgh EH8 9XD, Scotland, UK.
Mohamad Y
MY
Loqman
Peter G
PG
Bush
Colin
C
Farquharson
Andrew C
AC
Hall
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D027321:Anion Transport Proteins / Q000378:metabolism*; D017920:Antiporters / Q000378:metabolism*; D001846:Bone Development / Q000187:drug effects*; D001842:Bone and Bones / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D019902:Chondrocytes / Q000187:drug effects / Q000378:metabolism; D006132:Growth Plate / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D049990:Membrane Transport Modulators / Q000037:antagonists & inhibitors / Q000378:metabolism; D008682:Metatarsal Bones; D009924:Organ Culture Techniques; D010012:Osteogenesis / Q000187:drug effects* / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D064606:SLC4A Proteins; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*
D027321:Anion Transport Proteins; D017920:Antiporters; D049990:Membrane Transport Modulators; D064606:SLC4A Proteins; C452850:Slc9a1 protein, rat; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers; D000584:Amiloride; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; C039614:ethylisopropylamiloride