Ca2+‐dependent down‐regulation of human histamine H1 receptors in Chinese hamster ovary cells

G_q/11 protein‐coupled human histamine H₁ receptors in Chinese hamster ovary cells stimulated with histamine undergo clathrin‐dependent endocytosis followed by proteasome/lysosome‐mediated down‐regulation. In this study, we evaluated the effects of a sustained increase in intracellular Ca²⁺ concentrations induced by a receptor‐bypassed stimulation with ionomycin, a Ca²⁺ ionophore, on the endocytosis and down‐regulation of H₁ receptors in Chinese hamster ovary cells. All cellular and cell‐surface H₁ receptors were detected by the binding of [³H]mepyramine to intact cells sensitive to the hydrophobic and hydrophilic H₁ receptor ligands, mepyramine and pirdonium, respectively. The pretreatment of cells with ionomycin markedly reduced the mepyramine‐ and pirdonium‐sensitive binding sites of [³H]mepyramine, which were completely abrogated by the deprivation of extracellular Ca²⁺ and partially by a ubiquitin‐activating enzyme inhibitor (UBEI‐41), but were not affected by inhibitors of calmodulin (W‐7 or calmidazolium) and protein kinase C (chelerythrine or GF109203X). These ionomycin‐induced changes were also not affected by inhibitors of receptor endocytosis via clathrin (hypertonic sucrose) and caveolae/lipid rafts (filipin or nystatin) or by inhibitors of lysosomes (E‐64, leupeptin, chloroquine, or NH₄Cl), proteasomes (lactacystin or MG‐132), and a Ca²⁺‐dependent non‐lysosomal cysteine protease (calpain) (MDL28170). Since H₁ receptors were normally detected by confocal immunofluorescence microscopy with an antibody against H₁ receptors, even after the ionomycin treatment, H₁ receptors appeared to exist in a form to which [³H]mepyramine was unable to bind. These results suggest that H₁ receptors are apparently down‐regulated by a sustained increase in intracellular Ca²⁺ concentrations with no process of endocytosis and lysosomal/proteasomal degradation of receptors.

     

Isolation and characterization of a fibrogenic factor from CCl4-damaged rat liver

A fibrogenic factor which stimulates collagen production without cell proliferation of rat skin fibroblast cultures was isolated from CCl₄-damaged rat liver. (1) The factor was isolated from saline extracts of CCl₄-induced fibrotic rat liver and fractionated by Sephadex G-50S gel filtration and DEAE-cellulose chromatography. The original extract produced a 6-fold increase in collagen synthesis and the active factor eluted from gel filtration columns in a region corresponding to 5000 daltons. (2) The active factor was destroyed by heat (57°C, 30 min), phospholipase C digestion, but was insensitive to proteolytic enzymes or phospholipase A. Chemical analysis of the partially purified factor revealed relatively high quantities of phosphorus (3%) and low quantities of protein (13.3%), neutral sugar (1.9%) and uronic acid (4.9%). The possibility of this component being a complex phospholipid containing polypeptide is suggested. (3) Fibrogenic properties of the isolated factor was enhanced by apparent oxidation in air, to a more active, yet insoluble complex. Attempts to solubilize the oxidized product completely destroyed its biological activity.     

Procollagen production by rat hepatocytes in primary culture

Hepatocytes were obtained from rat liver and maintained in primary culture for periods up to 14 days. Collagen synthesis was maximal after 3–5 days and declined thereafter. The rate of collagen production was appox. one-tenth that observed by the rat skin fibroblasts of the same animals after 3–5 passages. Type I procollagen, the major macromolecular collagenous species, was identified as a 450 000 dalton molecule which was converted to 120 000 dalton, denatured, reduced procollagen chains. Prior pepsin digestion of the native procollagen released 95 000 dalton collagen chains identified as α1(I) and α2(I) by co-migration with carrier rat skin type I collagen chains. The production of type III procollagen was also tentatively identified by DEAE-cellulose chromatography. This material was isolated and identified with type-specific antibodies developed against the amino-terminal extension peptide of bovine skin type III procollagen. The relative distribution of type I:type III procollagen was estimated at 7:3 similar to the ratio previously found in whole rat liver. No evidence of type IV or type V procollagen biosynthesis was observed. These results suggest that rat hepatocytes in primary culture are capable of interstitial type I and type III collagen biosynthesis in a ratio similar to that found in their parent hepatic tissue in situ. They also suggest that the less abundant type IV (basement membrane-associated) or type V are nor major collagenous products of these cells.