Intravascular haemolysis is a physiological phenomenon as well as a severe pathological complication when accelerated in various autoimmune, infectious (such as malaria) and inherited (such as sickle cell disease) disorders. Haemoglobin released into plasma is captured by the acute phase protein haptoglobin, which is depleted from plasma during elevated haemolysis. Here we report the identification of the acute phase-regulated and signal-inducing macrophage protein, CD163, as a receptor that scavenges haemoglobin by mediating endocytosis of haptoglobin-haemoglobin complexes. CD163 binds only haptoglobin and haemoglobin in complex, which indicates the exposure of a receptor-binding neoepitope. The receptor-ligand interaction is Ca2+-dependent and of high affinity. Complexes of haemoglobin and multimeric haptoglobin (the 2-2 phenotype) exhibit higher functional affinity for CD 163 than do complexes of haemoglobin and dimeric haptoglobin (the 1-1 phenotype). Specific CD163-mediated endocytosis of haptoglobin-haemoglobin complexes is measurable in cells transfected with CD163 complementary DNA and in CD163-expressing myelo-monocytic lymphoma cells.

I25I-insulin binding and receptor-mediated insulin degradation were studied in isolated human fat cells from subcutaneous tissue. A high albumin concentration during cell isolation and incubation protected the fragile human adipocyte from lysis. Binding of tracer was pH dependent with an optimum between 7.4 and 7.6. At 37~ steady state was reached by 45 min and maintained for at least 2 h. The binding of labelled insulin in the presence of 10 ~tmol/l unlabelled insulin was only 1-4% of the total insulin binding. The half-maximal displacement of tracer iodoinsulin (10 pmol/1) by unlabelled insulin occurred at 0.25 nmol/l. Kinetic studies of the dissociation of labelled iodoinsulin from fat cells showed a slight acceleration in the presence of a high concentration of unlabelled insulin in the washout buffer as compared to a buffer containing no insulin. At steady state binding about 95% of the cell-associated radioactivity was extracted as iodoinsulin as judged by gel filtration. The remaining 5% co-eluted with iodotyrosine. During 60 rain about 90% of the cellassociated radioactivity dissociated as iodoinsulin and the rest as iodotyrosine. Conclusions: 1) A high albumin content of buffers prevents traumatization of the human adipocyte; 2)under these conditions steady state binding of insulin is readily measured at 37 ~ 3) the use of a washing procedure makes the non-specific binding negligible; 4) the human adipocyte insulin receptor has a very high affinity; 5) receptor-mediated insulin degradation is minimal.

Primary monolayer cultures of rat hepatocytes were used for studies of long-term and acute effects of hormones on the cyclic AMP system. When hepatocyte lysates were assayed at various times after plating of the cells three major changes in the metabolism of cyclic AMP and its regulation were observed : Glucagon-sensitive adenylate cyclase activity gradually declined in culture. In contrast, catecholamine-sensitive activity, being very low in normal adult male rat liver and freshly isolated hepatocytes, showed a strong and rapid increase after seeding of the cells. Concomitantly, there was an early elevation (peak z 6 h) and a subsequent decrease in activity of both high-& and low-K, cyclic AMP phosphodiesterase. These enzymic changes probably explained the finding that in intact cultured cells the cyclic AMP response to glucagon was diminished for 2-24 h after seeding, followed by an increase in the responsiveness to glucagon as well as to adrenergic agents up to 48 h of culture. Supplementation of the culture media with dexamethasone and/or insulin influenced the formation and breakdown of cyclic AMP in the hepatocytes. Insulin added at the time of plating moderately increased the adenylate cyclase activity assayed at 48 h, while dexamethasone had no significant effect. In the presence of dexamethasone, insulin exerted a stronger, and dosedependent (1 pM-1 pM), elevation of the adenylate cyclase activity in the lysates, particularly of the glucagon responsiveness. Thus, insulin plus dexamethasone counteracted the loss of glucagon-sensitive adenylate cyclase activity occurring in vitro. Kinetic plots of the cyclic AMP phosphodiesterase activity showed three affinity regions for the substrate. Of these, the two with high and intermediate substrate affinity (K, x 1 and x 10 pM) were decreased in the dexamethasone-treated cells. Insulin partly prevented this effect of dexamethasone. Accumulation of cyclic AMP in intact cells in response to glucagon or P-adrenergic agents was strongly increased in cultures pretreated with dexamethasone. The results suggest that insulin and glucocorticoids modulate the effects of glucagon and epinephrine on hepatocytes by exerting long-term influences on the cyclic AMP system. Hepatocytes maintained in vitro as primary cultures [I -101 offer an attractive experimental tool, not only due to the potential usefulness of these cells in investigations of the biology and pharmacology of liver [ll-131, but also because relatively few other model systems exist for long term studies of differentiated epithelial cells in vitro under controlled conditions.We have investigated the cyclic AMP system and its regulation in primary monolayer cultures of adult rat hepatocytes. The studies first intended to explore if the hepatocytes in culture maintain normal formation and degradation of cyclic AMP. Hepatocytes in monolayer possess the enzymes involved in cyclic AMP metabolism [5,, but relatively few details are known. Our results indicate that the cells in culture retain hormone sensitivity, but al...

Insulin binding, initial velocity of [14C]methylglucose transport, uptake of [14C]deoxyglucose and conversion of [U-14C]glucose to CO2, glyceride-glycerol and fatty acids were measured at 37 degrees C in adipocytes from rats of different weights (135-450 g) and therefore with different mean cell volumes (53-389 pl). Insulin binding per cell increased with increasing cell size and binding was 2.3 times higher in the largest cells than in the smallest cells with tracer alone. The difference was largely accounted for by an increase in the apparent affinity. Influx of methylglucose per cell increased with increasing cell size in the absence of insulin and remained constant as a function of cell size in its presence. The effect of insulin ranged from 11 fold in small cells to 3.5 fold in large cells. The rat of conversion of [U-14C]glucose to CO2 and lipids was about half of the rate of methylglucose transport under all conditions. In contrast, the uptake of deoxyglucose in insulin-stimulated cells decreased markedly with increasing cell size. Increasing cell size caused a small decrease in sensitivity which could be explained by a smaller amont of insulin bound per unit surface area. The results show that increasing cell size/animal weight causes changes in insulin binding which may explain changes in sensitivity. In addition, the hexose transport system is modified in a way which is not explained by changes in insulin binding. Finally, changes in deoxyglucose uptake with cell size do not parallel changes in methylglucose transport.