Effect of parathyroid hormone on amino acid transport by cultured neonatal mouse calvarial bone cells

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Abstract
The effect of synthetic bovine parathyroid hormone [bPTH-(1–34)] on amino acid uptake by confluent primary cultures of osteoblast-like cells isolated from neonatal mouse calvaria was studied. The uptake of proline and leucine by membrane transport Systems A, ASC, and L was discriminated on the basis of their sodium dependency and sensitivity to the system-specific amino acid analogs 2-(methylamino)-isobutyric acid (MeAIB) for System A and 2-amino-(2,2,1)-heptane-2-carboxylic acid (BCH) for System L. Treatment with 24 nM bPTH-(1–34) in serum-free EBSS for 4 hr increased the initial uptake rate of proline by 50–80% but had no effect on the uptake of leucine. Temporally, the increase in proline uptake was preceded by a 2-hr lag period and plateaued after 5–6 hr. A 5-min exposure to the hormone was sufficient to cause a significant increase in proline uptake measured 4 hr later. The magnitude of the increase was dose-related from 0.24 to 240 nM bPTH-(1–34), with the half-maximal effect occurring at 2.4 nM. Only the sodium-dependent, MeAIB-inhibitable component of proline uptake was elevated. Eadie-Hofstee analysis indicated that bPTH-(1–34) increased Vmax without changing the Km. Actinomycin D and cycloheximide prevented the hormone-stimulated increase, suggesting that RNA and protein synthesis were required. Treatment with either inhibitor alone caused a 30–35% decrease in proline transport that was not observed in the presence of bPTH-(1–34), indicating an effect not dependent on macromolecular synthesis. DBcAMP also increased proline uptake. Maximally effective concentrations of bPTH-(1–34) and DBcAMP did not increase transport above the level of bPTH-(1–34) alone. These results confirm that PTH and DBcAMP increase amino acid uptake in bone. In addition, they (1) identify osteoblast-like cells as targets for this action of PTH, (2) demonstrate that the effect is on amino acid transport System A, (3) suggest that the rise in proline uptake is a direct action of PTH that may involve effects on both synthesis and degradation of System A transport proteins, and (4) indicate that the effect may be mediated by cAMP.
Funding Information
  • National Institute on Aging (AG-02731)

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