Factor D (D) is an essential component of the alternative complement pathway. To determine whether D is catabolized by the kidney and, if so, at what site, we studied the renal handling of human D by in vivo nephron microperfusion and in vitro perfusion of rat kidneys. Human D was purified and labeled with 125I. Individual nephrons were perfused in vivo at varying rates with perfusate that contained 125I-D and [14C]inulin. When nephrons were perfused from proximal sites with perfusate 125I-D in a concentration of 3.0 micrograms/ml, urinary recovery of 125I-D increased (P less than 0.05) from 57.7 +/- 5.0 to 74.4 +/- 2.5% as tubule fluid flow rate was increased from 10 to 40 nl/min; recovery of 125I-D was less than (P less than 0.001) [14C]inulin recovery at all perfusion rates. At 20 nl/min, an increase in perfusate 125I-D concentration from 1.5 to 3.0 micrograms/ml was associated with an increase (P less than 0.001) in urinary 125I-D recovery (42.1 +/- 4.0 vs. 65.8 +/- 2.6%). Similarly, the addition of unlabeled D, 30 micrograms/ml, to 125I-D, 3.0 micrograms/ml, increased urinary 125I-D recovery (95.3 +/- 2.1%) at 20 nl/min. When nephrons were perfused from early distal segments at 10 nl/min, 125I-D recovery (91.2 +/- 4.3%) did not differ from [14C]inulin recovery (95.8 +/- 1.3%). In the isolated perfused filtering kidney, the concentration of intact 125I-D in the perfusate declined 60.3 +/- 14.6% over 1 h. 83.4 +/- 6.3% of the decrement in 125I-D was catabolized by the kidney; the remainder was excreted in the urine as intact D. When glomerular filtration was prevented by increasing perfusate albumin concentration to 16 g/dl, perfusate intact (125I-D) remained unchanged over 1 h. These data show that human D is catabolized by the kidney via glomerular filtration and reabsorption by the proximal nephron. Reabsorption of D appears to be a saturable process.
P W Sanders, J E Volanakis, S G Rostand, J H Galla
Histidine-rich glycoprotein (HRGP) is a human plasma and platelet protein of apparently diverse biological functions. In this study a new interaction for HRGP is described. HRGP specifically interacts with fibrinogen as demonstrated by two independent systems. Using an enzyme-linked immunosorbent assay it was demonstrated that HRGP bound to adsorbed fibrinogen in a concentration-dependent and saturable manner, with an apparent dissociation constant (Kd) of 6.7 nM. The binding was specific, reversible, and not mediated by a conformationally altered adsorbed fibrinogen molecule. The interaction was divalent cation-dependent and ionic in nature. The HRGP-fibrinogen interaction was also demonstrated using rocket immunoelectrophoresis. The HRGP-fibrinogen interaction had an effect on the kinetics of conversion of fibrinogen to fibrin as demonstrated by a prolongation of the thrombin time. HRGP also became incorporated into fibrin clots in a concentration-dependent and saturable manner, with an apparent Kd of 0.25 microM. The incorporation of HRGP into fibrin clots occurred in a plasma milieu as demonstrated by the direct incorporation of radiolabeled HRGP into plasma clots and by a significant decrease in serum HRGP levels as compared with plasma levels. HRGP prolonged the lag time phase of fibrin gel formation, and decreased the rate of turbidity rise, as well as the final absorbance of fibrin gels. Since the extent of fibrin polymerization was not influenced by the presence of HRGP, these data suggest that fibrin is distributed over more, but thinner, fibrils in the presence of HRGP. In addition to its potential effect on fibrin polymerization, the HRGP-fibrin interaction may play a role in the cell-cell interactions of platelets and macrophages.
L L Leung
To determine the biochemical basis of the oxidant-induced injury of cells, we have studied early changes after exposure of P388D1 murine macrophages to hydrogen peroxide. Total intracellular NAD+ levels in P388D1 cells decreased with H2O2 concentrations of 40 microM or higher. Doses of H2O2 between 0.1 and 2.5 mM led to an 80% depletion of NAD within 20 min. With doses of H2O2 of 250 microM or lower, the fall in NAD and, as shown previously, ATP, was reversible. Higher doses of H2O2 that cause ultimate lysis of the cells, induced an irreversible depletion of NAD and ATP. Poly-ADP-ribose polymerase, a nuclear enzyme associated with DNA damage and repair, which catalyzes conversion of NAD to nicotinamide and protein-bound poly-ADP-ribose, was activated by exposure of the cells to concentrations of 40 microM H2O2 or higher. Activation of poly-ADP-ribose polymerase was also observed in peripheral lymphocytes incubated in the presence of phorbol myristate acetate-stimulated polymorphonuclear neutrophils. Examination of the possibility that DNA alteration was involved was performed by measurement of thymidine incorporation and determination of DNA single-strand breaks (SSB) in cells exposed to H2O2. H2O2 at 40 microM or higher inhibited DNA synthesis, and induced SSB within less than 30 s. These results suggest that DNA damage induced within seconds after addition of oxidant may lead to stimulation of poly-ADP-ribose polymerase, and a consequent fall in NAD. Excessive stimulation of poly-ADP-ribose polymerase leads to a fall in NAD sufficient to interfere with ATP synthesis.
I U Schraufstatter, D B Hinshaw, P A Hyslop, R G Spragg, C G Cochrane
A multitracer stable isotope study of lysine kinetics was carried out in fasted adult female volunteers to determine whether a multicompartmental model that partitions protein synthesis and breakdown into at least two types of tissue components can be constructed from plasma and breath data. Five female subjects, maintained on formula diets, received L-[13C1]lysine (27 mumol/kg) as an i.v. bolus and L-[15N2]lysine (27 mumol/kg) as an oral bolus 4 h postprandially. Plasma and breath samples were collected for 6 h. On an alternate day, subjects received NaH13CO3 (10 mumol/kg) as an i.v. bolus and breath samples were collected for 6 h. Plasma tracer lysine levels were determined by gas chromatography-mass spectrometry isotope ratiometry, and breath 13CO2 levels were measured by mass spectrometric gas isotope ratiometry. The tracer data could be fitted to a mammillary multicompartmental model that consisted of a lysine central compartment and slow- and fast-exchanging peripheral compartments containing 37, 38, and 324 mumol/kg, respectively. The rates of lysine oxidation, incorporation into protein, and release by protein breakdown were 21, 35, and 56 mmol/kg/h, respectively, in the fast-exchanging compartment, whereas the rates of protein synthesis and breakdown in the slow compartment were both 53 mmol/kg/min. These values corresponded to a whole-body lysine flux of 106 mmol/kg/h. The kinetic parameters were in excellent agreement with reported values obtained by constant-infusion methods. The measurements indicated that it will be possible to detect changes in amino acid pool sizes and protein synthesis and breakdown associated with the mobilization of protein stores from plasma and breath measurements in multitracer stable isotope experiments.
C S Irving, M R Thomas, E W Malphus, L Marks, W W Wong, T W Boutton, P D Klein
Adherence to a substratum is a characteristic feature of monocyte-macrophages which may be required for several effector functions. Human peripheral blood monocytes selected by adherence were found to readhere preferentially at 1 h to fibronectin or to a biological matrix. There was then a progressive decrease in the number of adherent cells, and by 48 h only 8-20% of monocytes remained adherent. This loss of adherence occurred while monocytes remained viable by criteria such as exclusion of trypan blue or release of lactate dehydrogenase. 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) maintained the adherence of cultured monocytes to tissue culture plastic as well as to the biological matrix. This effect was concentration- and time-dependent, and suppressed by inhibitors of protein synthesis. Cellular proteins were labeled after incubation with [35S]methionine. Analysis by two-dimensional gel electrophoresis revealed increased labeling of several distinct proteins in 1,25-(OH)2D3-treated monocytes compared with control monocytes. The increased loss of adherence and decreased overall protein synthesis observed in monocytes incubated at 45 degrees C was partially prevented by preincubation of the cells with 1,25-(OH)2D3. We further evaluated the effects of thermal stress and 1,25-(OH)2D3 on protein synthesis by monocytes, and found that 1,25-(OH)2D3 increased the synthesis of heat shock proteins, protected normal protein synthesis, and increased the rate of recovery of normal protein synthesis after the thermal stress. These observations suggest that 1,25-(OH)2D3 influences monocytes by preserving the synthesis of proteins, including those critical for the maintenance of cell adherence.
B S Polla, A M Healy, E P Amento, S M Krane
The capsular polysaccharide of Hemophilus influenzae type b, polyribosyl ribitol phosphate (PRP), is released from growing organisms during human infection and can be found in body fluids. It binds to untreated erythrocytes. Many patients with invasive infections with this organism develop significant hemolysis, but the mechanism has been unclear. We have found that PRP binds to human erythrocytes in vivo. PRP-coated erythrocytes have a shortened circulation time in mice, but do not lyse spontaneously or fix complement. PRP-coated erythrocytes exposed to antiserum to H. influenzae type b are undamaged in the absence of complement, but are rapidly and effectively lysed in the presence of an intact complement system both in vitro and in vivo in mice. PRP-coated red cells are taken up by liver and spleen. Antiserum to PRP increases hepatic uptake of PRP-coated red cells more than splenic, and appears to induce intravascular, complement-mediated hemolysis, as well as extravascular hemolysis. Patients with invasive infection develop hemolysis when circulating PRP and antibody to PRP are present simultaneously. PRP can sometimes be detected on patient erythrocytes when free PRP is present in serum, but this is an inconsistent finding. The hemolytic anemia that occurs during human infection with H. influenzae type b may be due to absorption of PRP to red cells and immune destruction of sensitized erythrocytes. The process requires an intact complement system; both complement-mediated cell lysis and extravascular hemolysis contribute to red cell destruction.
S B Shurin, P Anderson, J Zollinger, R K Rathbun
Calcium has been proposed as an intracellular second messenger for activation of secretion, phagocytosis, and the oxidative burst of neutrophils. We have examined the role of calcium in human monocyte activation. Concanavalin A (Con A)-stimulated monocytes displayed an increment in cytoplasmic ionized calcium at 31 +/- 6 s and the onset of superoxide production at 61 +/- 9 s. The increase in cytoplasmic calcium invariably preceded the onset of superoxide production. If the external calcium concentration was reduced to less than 28 nM by the addition of 10 mM EGTA, superoxide production was not diminished at 5 min; however, superoxide production decreased thereafter. The Con A-evoked increment in cytoplasmic ionized calcium was blunted upon the addition of EGTA and decreased further with time. Both the production of superoxide and the Con A-evoked increment in cytoplasmic ionized calcium displayed a 50% inhibition after 15 min of calcium depletion and were completely inhibited after 60 min. Total cell calcium fell from 0.7 to 0.5 fmol/cell, and the basal level of ionized calcium fell from 83 to 30 nM after 60 min. Histidine, a strong chelator of divalent cations other than calcium and magnesium, had no effect on monocyte superoxide production or on ionized calcium concentrations, indicating that EGTA inhibition was due to cell calcium depletion. In calcium-depleted cells, Con A did not evoke superoxide production until calcium was restored to the incubation medium. The restoration of calcium to Con A-treated, calcium-depleted monocytes permitted a rapid rise in the cytoplasmic ionized calcium, and the production of superoxide within 9 s. These data suggest that an increase in ionized cytoplasmic calcium is necessary for the activation of monocyte superoxide production by Con A. The rise in ionized calcium in response to Con A results, in part, from an internal redistribution of calcium, which is sufficient to permit superoxide generation.
S P Scully, G B Segel, M A Lichtman
In conscious dogs, phentolamine infusion significantly increased fasting portal vein insulin, glucagon, and decreased net hepatic glucose output and plasma glucose. Propranolol significantly decreased portal vein insulin, portal flow, and increased hepatic glucose production and plasma glucose. Phentolamine, propranolol, and combined blockade reduced glucose absorption after oral glucose. alpha, beta, and combined blockade abolished the augmented fractional hepatic insulin extraction after oral glucose. Despite different absolute amounts of glucose absorbed and different amounts of insulin reaching the liver, the percent of the absorbed glucose retained by the liver was similar for control and with alpha- or beta blockade, but markedly decreased with combined blockade. Our conclusions are: (a) phentolamine and propranolol effects on basal hepatic glucose production may predominantly reflect their action on insulin and glucagon secretion; (b) after oral glucose, alpha- and beta-blockers separately or combined decrease glucose release into the portal system; (c) net hepatic glucose uptake is predominantly determined by hyperglycemia but can be modulated by insulin and glucagon; (d) direct correlation does not exist between hepatic delivery and uptake of insulin and net hepatic glucose uptake; (e) alterations in oral glucose tolerance due to adrenergic blockers, beyond their effects on glucose absorption, can be, to a large extent, mediated by their effects on insulin and glucagon secretion reflecting both hepatic and peripheral glucose metabolism.
Z Chap, T Ishida, J Chou, L Michael, C Hartley, M Entman, J B Field
We have examined whether the toxic effects of homocysteine on cultured endothelial cells could result from the formation and action of hydrogen peroxide. In initial experiments with a cell-free system, micromolar amounts of copper were found to catalyze an oxygen-dependent oxidation of homocysteine. The molar ratio of homocysteine oxidized to oxygen consumed was approximately 4.0, which suggests that oxygen was reduced to water. The addition of catalase, however, decreased oxygen consumption by nearly one-half, which suggests that H2O2 was formed during the reaction. Confirming this hypothesis, H2O2 formation was detected using the horseradish peroxidase-dependent oxidation of fluorescent scopoletin. Ceruloplasmin was also found to catalyze oxidation of homocysteine and generation of H2O2 in molar amounts equivalent to copper sulfate. Finally, homocysteine oxidation was catalyzed by normal human serum in a concentration-dependent manner. Using cultured human and bovine endothelial cells, we found that homocysteine plus copper could lyse the cells in a dose-dependent manner, an effect that was completely prevented by catalase. Homocystine plus copper was not toxic to the cells. Specific injury to endothelial cells was seen only after 4 h of incubation with homocysteine plus copper. Confirming the biochemical studies, ceruloplasmin was also found to be equivalent to Cu++ in its ability to cause injury to endothelial cells in the presence of homocysteine. Since elevated levels of homocysteine have been implicated in premature development of atherosclerosis, these findings may be relevant to the mechanism of some types of chronic vascular injury.
G Starkebaum, J M Harlan
Pharmacologic doses of glucocorticoids are administered to patients with adrenal insufficiency during operative procedures to prevent hemodynamic instability, cardiovascular collapse, and death. Since these supraphysiologic doses might not be necessary and might have adverse effects, we examined the effects of different doses of glucocorticoids on hemodynamic adaptation during surgical stress in adrenalectomized primates. Sham-adrenalectomized placebo-treated animals served as controls. Adrenalectomized monkeys were maintained for 4 mo on physiologic glucocorticoid and mineralocorticoid replacement. The adrenalectomized monkeys were then stratified into three groups receiving, respectively, subphysiological (one-tenth the normal cortisol production rate), physiological, or supraphysiological (10 times the normal cortisol production rate) cortisol (hydrocortisone) treatment. 4 d later a cholecystectomy was performed. The intraoperative hemodynamic and metabolic parameters, perioperative survival rates, and postoperative wound healing were compared. The subphysiologically treated group was hemodynamically unstable before, during, and after surgery and had a significantly higher mortality rate than control. In this group, arterial blood pressure was low, and the cardiac index, systemic vascular resistance index, and left ventricular stroke work index were all reduced, suggesting decreased cardiac contractility and blood vessel tone. In contrast, the physiologically replaced group was indistinguishable from either supraphysiologically treated animals or sham-operated controls. All groups had similar metabolic profiles and normal wound healing. These findings suggest that the permissive actions of physiologic glucocorticoid replacement are both necessary and sufficient for primates to tolerate surgical stress. Supraphysiological glucocorticoid treatment has no apparent advantage during this form of stress in the primate.
R Udelsman, J Ramp, W T Gallucci, A Gordon, E Lipford, J A Norton, D L Loriaux, G P Chrousos