Suppression of mammalian bone growth by membrane transport inhibitors

Abstract

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.

Cysteine induces longitudinal bone growth in mice by upregulating IGF-I

“…Longitudinal bone growth is a results of the proliferation, hypertrophy and matrix production of cartilage cells in growth plates (1). The growth plate is a relatively thin layer of cartilage which is found in growing long bones during longitudinal skeleton growth (2,3). The growth of the long bone at the growth plate is regulated by an endocrine signaling systems that involves glucocorticoids, growth hormone (GH) and insulin-like growth factor-I (IGF-I) (4).…”

A simple method based on confocal microscopy and thick sections recognizes seven subphases in growth plate chondrocytes

“…One major factor that determines ion mobility and the net movement of water into the chondrocyte is the interaction with an ionic microenvironment with high cationic content due to the concentration of fixed negative charges on proteoglycans of the cartilage extracellular matrix. Interactions between transported molecules with the microenvironment and both active and passive membrane transport proteins have been documented in cell volume regulation of chondrocytes 4,5 . Igf1 expression was in the clusters 1 to 4 and a significant increase occurred in the transition from cluster 4 to cluster 5.…”

“…However, information regarding how the cell volume increase occurs is relatively scarce. Hypertrophic chondrocytes have great amount of cytoplasm with sparse organelles and numerous membrane transporters, and volume increase is due to unbalanced water accumulation or swelling 3,4 as well as to synthesis of cytoplasmic components or true hypertrophy 5 . Increase of chondrocyte volume was found to result from three sequential stages by using tomographic phase microscopy 6 : two stages of true hypertrophy (phases 1 and 3) when chondrocytes increase their volume with active synthesis of cytoplasmic components separated by a stage of swelling (phase 2), when volume keeps increasing but synthesis ceases and the cytoplasm density decreases.…”

Postnatal ex vivo rat model for longitudinal bone growth investigations

“…The round chondrocytes become flattened, proliferate and are then organized into columns . The proliferative chondrocytes then cease to divide and differentiate into prehypertrophic chondrocytes (PHCs) before undergoing a significant increase in cell volume of about 20‐fold to become hypertrophic chondrocytes (HCs) . The HCs are considered to be specialized cells that signify the end‐state of the chondrocyte differentiation pathway, and play a crucial role during endochondral ossification .…”