ABSTRACT
We characterize here a lysosomal cystine transporter in mouse L-929 fibroblasts. Granular fractions from cells preloaded with cystine demonstrated countertransport that showed no dependence on Na+ or K+. The Michaelis constant for infinite-trans influx was 0.27 +/- 0.06 mM (n = 3), and a nonsaturable component of cystine entry was observed with Kd = 0.8-1.8 nmol of cystine.min-1.unit of hexosaminidase-1.mM-1. We found no evidence that cystine was also carried on any of the other known lysosomal amino acid transporters. Over 50 analogs were tested for their ability to inhibit countertransport. The inhibition constants are reported for selenocystine, cystathionine, selenomethionine, and leucine. Significant requirements for recognition by the transporter were the presence of amino groups, L configuration, and a chain length not greater than eight atoms. A net positive or negative charge was not required. Some di- as well as tetrapolar amino acids were recognized. We have surmised that the binding site has polar and apolar domains, the latter being large enough to accommodate branching on C-3 and the substitution of selenium or carbon in place of sulfur.
Subject(s)
Cystine/metabolism , Lysosomes/metabolism , Amino Acids/pharmacology , Animals , Biological Transport/drug effects , Computer Graphics , Cystine/analogs & derivatives , Cystine/pharmacology , Cytoplasmic Granules/metabolism , Kinetics , L Cells/metabolism , Lysosomes/drug effects , Mice , Models, Molecular , Molecular Conformation , beta-N-Acetylhexosaminidases/metabolismABSTRACT
The production of inflammatory mediators by glomerular cells may be instrumental in the development of pathophysiological alterations during glomerulonephritis. Since bacterial lipopolysaccharide (LPS) is a naturally occurring immunological stimulus, we studied its inflammatory effects on isolated renal glomeruli. LPS stimulation of human and rat isolated glomeruli resulted in a dose- and time-dependent platelet-activating factor (paf-acether) production. Maximal paf-acether generation (1.04 to 1.50 ng/mg protein) (n = 18) was obtained when glomeruli were stimulated for periods of 1 to 4 hr and with 1-2 micrograms/ml LPS. Paf-acether derived from human and rat glomeruli exhibited identical biological and physicochemical characteristics. In addition, rat glomeruli stimulated with doses of LPS from 100 ng to 50 micrograms/ml released an Interleukin-1 (IL-1)-like cytokine differing in part from that described in cultured mesangial cells. Maximal release of IL-1-like activity by rat glomeruli was obtained after 24 to 48 hr incubation in the presence of LPS. After gel chromatography resolution, the glomerular cytokine presented IL-1-like activity in fractions corresponding to molecular weights of 15-35 and 4-8 kDa. The latter compounds could represent metabolites similar to those described in normal urine. Thus the local release of paf-acether and IL-1-like cytokine by glomeruli in response to bacterial stimuli may represent a prominent feature of glomerular inflammation.
