The long-term objective of this proposal is to elucidate the mechanisms which regulate the secretion of the rabbit acute phase protein, C-reactive protein (CRP) by hepatocytes. Studies of CRP synthesis and secretion by primary hepatocyte cultures prepared from rabbits following in vivo inflammatory stimulus have indicated the existence of a dynamic intracellular CRP pool, the size of which is regulated during the course of the acute phase response (APR).
Specific aims of this proposal are 1) to determine the kinetics of turnover of intracellular CRP in hepatocyte cultures prepared from animals at varying stages of the APR, 2) to identify the subcellular localization of the intravellular CRP pool and its relationship to CRP synthesis and secretion and 3) to determine whether newly-synthesized CRP is bound to lipoprotein prior to its secretion. Specifically, we will study the kinetic of pulse-chase labelling of intracellular CRP to determine the extent, if any, of intracellular degradation of CRP. Under conditions of continuous labelling, analysis of the kinetics with which intra- and extracellular labelled CRP reach steady-state will serve to define the nature of the intracellular CRP pool (e.g., ordered vs randon turnover, number of pools). The possibililty that some CRP may exist in a kinetically inactive, storage pool will be investigated by incubation of cells with amino acids containing heavy, stable isotopes. Newly-synthesized CRP (heavy-labelled) will be distinguished from CRP which was present prior to addition of label by means of isopycnic centrifugation; both forms will be quantitated by radioimmunoassay (RIA). Subcellular localization of the CRP pool will be investigated by preparing endoplasmic reticulum, Golgi, and coated vesicle fractions from rabbit livers. Relative amounts of CRP and albumin in each fraction will be determined by RIA. Pulse-radiolabelled intracellular CRP and albumin, synthesized by cultured hepatocytes, will be quantitated by immunoprecipitation of each fraction. Since CRP present in serum has been reported to be bound to lipoproteins, we will determine whether CRP secreted by hepatocytes cultured in the absence of serum is bound to lipoprotein. The serum concentration of CRP is markedly elevatd in a number of inflammatory diseases and following many forms of tissue injury. Investigation of mechanisms which control CRP secretion will contribute to our understanding of the host response to a wode variety of acute and chronic inflammatory conditions, including many of the rheumatic diseases
| Hu, S I; Macintyre, S S; Schultz, D et al. (1988) Secretion of rabbit C-reactive protein by transfected human cell lines is more rapid than by cultured rabbit hepatocytes. J Biol Chem 263:1500-4 |
| Macintyre, S S (1988) C-reactive protein. Methods Enzymol 163:383-99 |
