Premium
Effect of parathyroid hormone on amino acid transport by cultured neonatal mouse calvarial bone cells
Author(s) -
Yee John A.
Publication year - 1988
Publication title -
journal of bone and mineral research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.882
H-Index - 241
eISSN - 1523-4681
pISSN - 0884-0431
DOI - 10.1002/jbmr.5650030214
Subject(s) - proline , parathyroid hormone , cycloheximide , leucine , amino acid , endocrinology , medicine , calvaria , chemistry , sodium , hormone , biology , biochemistry , protein biosynthesis , calcium , in vitro , organic chemistry
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 n M 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 n M bPTH‐(1–34), with the half‐maximal effect occurring at 2.4 n M. Only the sodium‐dependent, MeAIB‐inhibitable component of proline uptake was elevated. Eadie‐Hofstee analysis indicated that bPTH‐(1–34) increased V max without changing the K m . 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.