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Rabbit prekallikrein (RPK) was purified from rabbit plasma by ion exchange and lectin column chromatography and preparative polyacrylamide gel electrophoresis. A 1500-fold purification was routinely achieved with a final yield of 5–10%, The purified RPK was found to be a glycoprotein with an apparent molecular weight of 88,000. Activation of RPK with either trypsin or rabbit Hageman factor (active) occurs by limited proteolytic cleavage, producing two disulfide-linked polypeptide chains with molecular weights of 55,000 and 35,000. Both chains contain carbohydrate and the 35,000-molecular-weight polypeptide was shown to incorporate [3H]DFP. Activation of RPK in kaolin-treated plasma was shown to proceed by an analogous mechanism yielding 55,000- and 35,000-molecular-weight polypeptide chains.

Experiments were performed to probe the role of exocytotic and endocytotic processes in the regulation of the human granulocyte O-2-generating system. Analytical subcellular fractionation studies indicated that 25-30% of the total cellular b-cytochrome and 8-10% of the flavin co-sedimented with plasma membrane markers, irrespective of stimulation of the cells by the chemoattractants N-formyl-Met-Leu-Phe (FMLP) or C5a. Phorbol myristate acetate stimulation resulted in significant translocation of b-cytochrome but not flavin from the specific granule/Golgi to the plasma membrane-enriched fractions. These results indicated that approximately 3.1 X 10(5) flavin and 0.8-1 X 10(6) b-cytochrome molecules are present in the plasma membrane of an isolated unstimulated human granulocyte and that these levels are invariant upon stimulation with chemoattractants. Maximal instantaneous rates of O-2 generation by cells in these preparations, however, were equivalent for all the stimuli. Since stimulation of granulocytes by phorbol myristate acetate, FMLP, or C5a results in exocytosis and/or endocytosis, then the role of these processes in regulating stimulated O-2 production by controlling the content of plasma membrane redox enzymes is questionable. This conclusion was supported by observations made with cytoplasts, which do not have an intracellular reserve of granules. Cytoplasts prepared from granulocytes produced O-2 at equivalent rates as their parent cells on a per unit surface area basis. These results suggest: 1) that stimulation of granulocytes with chemotactic peptides leads to full generation of O-2 at the cell surface without exocytotic recruitment of additional b-cytochrome and flavoprotein from the cytoplasmic compartment; 2) that these redox enzymes are not internalized along with chemoattractant receptors; and 3) that traffic of these redox enzymes between endo- and plasma membranes is not involved in the regulation of O-2 production in suspensions of human granulocytes stimulated by chemoattractants.

In order to investigate the physicochemical properties of the N-formyl peptide receptor of human neutrophils, the receptor was specifically and covalently labeled with an iodinated, photoactivatable derivative of the chemotactic hexapeptide, N-formyl-norleucylleucyl- phenylalanyl-norleucyl-[125I]iodotyrosyl-N epsilon-(6- (4‘-azido-2‘-nitrophenylamino) hexanoyl)-lysine. After labeling isolated neutrophil membranes, the receptor was extracted with Triton X-100, digitonin, or octyl glucoside and subjected to gel filtration on a calibrated Ultrogel AcA 34 column. The Triton X-100- and digitonin-extracted receptor eluted as single molecular species, with Stokes radii of 40 and 33 A, respectively. This material was subjected to further physicochemical analysis. When octyl glucoside-extracted material was gel-filtered, a second peak containing specifically labeled material eluted in the void volume. Subsequent sodium dodecyl sulfate-polyacryl-amide amide gel electrophoresis analysis indicated that this species was the result of disulfide bonded aggregates containing the monomeric species. Sedimentation equilibrium analysis was carried out in H2O and D2O/H2O mixtures, yielding an apparent molecular mass of 63,000 daltons for both Triton X-100- and digitonin-extracted receptor. This agrees closely with the reduced sodium dodecyl sulfate-polyacrylamide gel electrophoretic value of 50,000-60,000 daltons, indicating that the receptor extracted from unstimulated membranes is monomeric in these detergents. From the sedimentation equilibrium data, the partial specific volume (v) and frictional ratio (f/f0) were calculated. The v is high in both Triton X-100 (0.880) and digitonin (0.829), indicating that the receptor may be associated with tightly bound endogenous lipid or that it is a hydrophobic membrane protein. This latter likelihood is further supported by the quantitative extraction of receptor into Triton X-114 by a phase-separation method. The frictional ratio of 1.1-1.3 is consistent with an elongated globular protein having an axial ratio of approximately 3:1. This in conjunction with the Stokes radius of 40 A would indicate that the receptor is capable of spanning the 35-40-A nonpolar center of the lipid bilayer. The state of the receptor in situ is discussed.

Inhibition of ADP phosphorylation by both glycolysis and mitochondria in P388D1 cells exposed to H2O2 is described. Net glucose uptake and lactate production were inhibited by oxidant exposure (ED50 = 50-100 microM). Glycolysis was specifically inactivated at the glyceraldehyde-3-phosphate dehydrogenase step by three independent mechanisms: (a) direct inactivation of the intracellular enzyme (ED50 approximately equal to 100 microM); (b) reduction of the intracellular concentration and redox potential of its nicotinamide cofactors; and (c) a cytosolic pH shift further from the enzyme optima. Consistent with inhibition of glycolysis at the glyceraldehyde-3-phosphate dehydrogenase step, a rise in the intracellular concentration of glyceraldehyde 3-phosphate, dihydroxyacetone phosphate, and fructose 1,6-bisphosphate was observed. The calculated combined inhibition of glyceraldehyde-3-phosphate dehydrogenase activity could be reasonably correlated with the depression in glycolytic flux rate with the appropriate modeling. The steady-state contribution by mitochondria to the total intracellular ATP pool was indirectly determined by the use of various metabolic inhibitors and was found to rapidly decline following exposure to 300-800 microM H2O2. The inhibition of ADP phosphorylation appeared to be related more to the direct inhibition of the ATPase-synthase complex rather than to the diminished capacity of the respiratory chain for coupled electron transport. Both the estimated rates of ADP phosphorylation by glycolysis and mitochondria and the estimated rate of ATP hydrolysis by ongoing metabolism were utilized to model the approximate decline in intracellular ATP expected at 15-min exposure to various H2O2 concentrations. Theoretical calculations and the measured intracellular ATP status were in good agreement. Oxidant exposure for 15 min resulted in dose-dependent killing of the cells (ED50 = 500 microM), indicating a close correlation between H2O2-mediated loss of intracellular ATP and cell viability. The possible contribution of impaired energy homeostasis during oxidant-mediated injury to the process of cell dysfunction and death is discussed.

The effect of C3 depletion on the multiple pathophysiological changes produced by a lethal dose of Serratia marcescens lipopolysaccharide (LPS) was evaluated. The injection of this LPS into rabbits resulted in biphasic hypotensive changes and thrombocytopenia. These changes were characterized by an acute, transient decrease occurring within minutes after injection followed by a second more gradual decrease beginning 30 to 60 min post-LPS. Prior depletion of C3, with the anticomplementary protein from cobra venom (CoF), did not alter the extent of either the gradual hypotensive and platelet changes or the coagulative and metabolic changes when normal and C3-depleted rabbits were compared. Importantly, the lethal effects of S. marcescens LPS were not reduced by prior depletion of C3. Only the immediate, reversible thrombocytopenia and hypotension were abrogated by C3 depletion.

The mechanisms by which human high molecular weight kininogen (HMKrK) contributes to the surface-dependent activation of the Hageman factor systems have been studied. The ability of various mixtures of purified human Hageman factor (coagulation factor XII), HMrK, prekallikrein, and kaolin to activate coagulation factor XI was determined with factor XIa (activated factor XI) clotting assays. Hageman factor, HMrK and prekallikrein were required for maximal rates of activation of factor XI. A certain optimal mixture of purified Hageman factor, HMrK, prekallikrein, and kaolin gave the same rapid initial rate of activation of purified factor XI as an equivalent aliquot of factor XI-deficient plasma. This suggests that potent, surface-mediated activation of factor XI in plasma is explicable in terms of Hageman factor, HMrK, and prekallikrein. By studying separately some of the surface-dependent reactions involving Hageman factor, it was found that HMrK accelerated by at least an order of magnitude the following reactions: (i) the activation of factor XI by activated Hageman factor; (ii) the activation of prekallikrein by activated Hageman factor; and (iii) the activation of Hageman factor by kallikrein. Stoichiometric rather than catalytic amounts of HMrK gave optimal activation of factor XI. These results are consistent with the hypothesis that HMrK and Hageman factor form a complex on kaolin which renders Hageman factor more susceptible to proteolytic activation by kallikrein and which facilitates the action of activated Hageman factor on its substrate proteins, factor XI and prekallikrein.

In the contact phase of activation of the kinin-forming, intrinsic clotting, and fibrinolytic systems, high-molecular-weight kininogen acts as a cofactor for the activation of Factor XI, prekallikrein, and Hageman factor. One mechanism by which high-molecular-weight kininogen acts as a cofactor has been studied by using 125I-labeled Factor XI and prekallikrein in kaolin-activated normal human plasma and plasmas deficient in high-molecular-weight kininogen and Hageman factor. High-molecular-weight kininogen was found to be essential for normal binding and cleavage of both Factor XI and prekallikrein on the kaolin surface. Hageman factor was essential for cleavage but not for binding of Factor XI and prekallikrein to kaolin. In normal plasma 80% of the activated Factor XI remained surface-bound, whereas 80% of the kallikrein was not surface-bound. These findings are consistent with the hypothesis that, in the initial phase of contact activation, high-molecular-weight kininogen links both Factor XI and prekallikrein to the exposed surface where they are activated by surface-bound activated Hageman factor. Once activated, the Factor XI molecules remain localized at the site of activation, in contrast to the kallikrein molecules which are found largely in the surrounding plasma.

Antifluorescein antibody molecules were used to interrupt the stimulation of neutrophils by a fluoresceinated chemotactic peptide. From the results we construct a semiquantitative relationship among ligand-receptor interaction, the time course of cell triggering and response, and aspects of cellular adaptation. The interaction of the antibody with the free fluoresceinated peptide is complete within a few seconds and the peptide-antibody complex neither stimulates the cells nor inhibits subsequent stimulation by unlabeled peptide. When antibody is added to a cell suspension that has been stimulated with the fluoresceinated peptide, we observe that: (i) the apparent membrane depolarization response monitored by a fluorescent dye can be inhibited only if antibody is added within 30 sec of stimulation; (ii) the superoxide response can be inhibited even if antibody is added more than 1 min after stimulation and decays with an intrinsic half-life of about 12 sec; (iii) responses to a second dose of nonfluoresceinated peptide are enhanced if the antibody is added within 2 min of stimulation by the fluoresceinated peptide. These results suggest that different neutrophil responses depend in individual ways on the time course and extent of ligand binding to its receptor. A comparison of these data with the time course of binding permits an estimate of the number of receptors involved in these responses.

We have developed a continuous spectroscopic method to analyze the kinetics of elastase secretion by human neutrophils. We have used the elastase-specific substrate methylsuccinylalanylalanylprolylvalylmethylcoumarin amide (Castillo, M. J., Nakajima, K., Zimmerman, J., and Powers, J. C. (1979) Anal. Biochem. 99, 53-64), which liberates the fluorophore, aminomethylcoumarin, when cleaved by elastase. We find that secretion of elastase in cytochalasin B-treated cells is initiated within approximately 5 s of exposure of the cells to the fluoresceinated chemotactic peptide stimulus, N-formyl-norleucylleucylphenylalanylnorleucyltyrosyllysine-fluorescein, and that secretion is completed within 30 s. The kinetics of this response is only slightly dependent on the concentration of the stimulus. Up to 100% (approximately 0.5 pg/neutrophil) of the elastase can be released in a dose-dependent manner by stimulated cells. We have also used this fluoresceinated stimulus and an antibody to fluorescein (Sklar, L. A., Oades, Z. G., Jesaitis, A. J., Painter, R. G., and Cochrane, C. G. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 7540-7544) to probe the temporal relationship between the binding of stimulus to the receptors and the cellular response. We find that the entire response is elicited by the binding which occurs within the first 15 s after the addition of stimulus, regardless of the dose. We estimate that an occupancy of no more than 20% of the cellular receptors for the stimulus is required to evoke the optimal response.

Interleukin-8 (IL-8) has at least two binding regions for both the A and the B type IL-8 receptors. This study defines an important region between Cys7 and Cys50 that, together with the Glu4-Leu5-Arg6 sequence of the NH2 terminus, accounts for the high affinity binding of IL-8 to the IL-8 A receptor on leukocytes. Utilizing rabbit IL-8 that shares 82% sequence identity with human IL-8, but has 200-fold lower binding affinity for the IL-8 A receptor, residues of the human homologue were sequentially exchanged into the rabbit molecule. Replacement of rabbit His13 and Thr15 with Tyr13 and Lys15 of the human molecule converted the low affinity binding of the rabbit IL-8 to the high affinity binding of human IL-8 as shown by both competitive binding and by Ca2+ mobilization. As a corollary, replacement of the Tyr13 and Lys15 of the human IL-8 with His13 and Thr15 of the rabbit IL-8 reduced binding activity of this mutated human IL-8 200-fold. The site of interaction on the IL-8 receptor type A for the Tyr13 and Lys15 sequence was found to be in the NH2-terminal region of this receptor. A structural pattern of the binding between IL-8 and the A type IL-8 receptor is proposed.

Six suction-induced blister fluids obtained from five patients with hereditary angioedema (HAE) contained active kallikrein, whereas only two blister fluids obtained from eight normal volunteers contained small amounts of this activity. Kallikrein was present in large amounts of HAE blister fluids as assessed by its ability to liberate smooth-muscle-contracting activity from purified high molecular weight kininogen. It was inhibited by purified antibodies specific for plasma prekallikrein and also by purified C1 inhibitor, but not by antibodies specific for C1s. These observations suggest that activation of the Hageman-factor-dependent pathways occurs in the tissues of HAE patients, and once generated, active kallikrein persists in these tissues.

The dissemination of contact activation of plasma was examined by measuring the cleavage of Hageman factor (HF) molecules on two separate sets of kaolin particles, one of which contained all of the components of the contact activation system, HF, prekallikrein (PK) and high molecular weight kininogen (HMWK) in whole normal plasma, and the second set of particles containing only HF and HMWK, being prepared with PK-deficient plasma. After mixing of the particles, cleavage of HF on the second set of particles occurred at a rate similar to that occurring on the first set of particles. This indicated that rapid dissemination and burst of activity of the contact reaction takes place in fluid phase. A supernatant factor, responsibel for the dissemination of the contact reaction, was identified as kallikrein. A rapid appearance of cleaved PK (kallikrein) and HMWK on both the kaolin surface and in the supernate was observed. Within 40 s, > 70-80% of the PK and HMWK in the supernate was cleaved. On the surface, approximately 70% of each radiolabeled protein was cleaved at the earliest measurement. Cleavage of PK by activated HF occurred at least 17 times faster on the surface than in the fluid phase, as virtually no cleavage of PK occurred in fluid phase. Each molecule of surface-bound, activated HF was calculated to cleave at a minimum, 20 molecules of PK per minute. It is concluded that the contact activaton of plasma may be divided into three phases: (a) the reciprocal activation of a few molecules of zymogen HF and PK on the surface, with HMWK acting as cofactor to bring these molecules into apposition; (b) the rapid release of kallikrein into the fluid phase and the continued conversion of PK to kallikrein by each surface-bound molecule of activated HF; and (c) the activation by fluid-phase kallikrein of multiple surface-bound HF molecules, and the cleavage of multiple molecules of MHWK both in fluid phase and on the surface by the soluble kallikrein. The evidence suggests that steps b and c account for a great majority of the generation of contact activation of plasma.

We have studied the role of complement in lipopolysaccharide (LPS)-induced hypotension and disseminated intravascular coagulation (DIC) by comparing the effects of injection of three preparations of LPS from E. Coli 0111:B4, S. minnesota Re595, and S. marcescens. Injections of nonlethal doses of these LPS preparations into normal rabbits produced decreases in mean arterial blood pressure during a 5-h period. When rabbits treated with cobra venom factor (CoF) to deplete C3 were injected with the various LPS preparations, mean arterial pressures fell at a rate and extent essentially identical to that observed in normal rabbits. Rabbits genetically deficient in C6 also demonstrated LPS-induced hypotensive changes. Only minimal, or no changes in plasma C3 levels or serum CH50 values were detected in normal rabbits after LPS injection. Hypotensive changes were also induced in rabbits when complement was rapidly activated by intravenous injection of CoF. In contrast to the hypotension induced by LPS, the fall in arterial pressure associated with the consumption of complement was short lived and required the rapid consumption of considerable amounts of C3. The occurrence of DIC noted in normal rabbits injected with each preparation of LPS was not inhibited in either rabbits treated with cobra factor or in C6-deficient rabbits. The DIC was most pronounced after injection of Re595 and S. marcescens LPS. Injection of the various LPS preparations produced a rapid disappearance of circulating neutrophils and mononuclear cells, which occurred with the same kinetics and to the same extent in normal, CoF-treated, and C6-deficient rabbits. Injection of either Re595 LPS or S. marcescens LPS produced a biphasic disappearance of circulating 51Cr-platelets. In contrast, injection of 0111:B4 LPS affected only slightly the rate of disappearance of 51Cr-platelets. Depletion of C3 by cobra factor treatment had no effect on the disappearance of platelets in animals injected with 0111:B4. In marked contrast cobra factor treatment greatly reduced the initial rapid disappearance of platelets in rabbits injected with either Re595 or S. marcescens LPS, but had no effect in the secondary disappearance phase.

The responses of neutrophils to formyl peptides are initiated and in many cases achieve a maximal level prior to equilibrium receptor occupancy. In order to begin to understand the linkage between receptor occupancy and cell response we have used a pulsed binding procedure to analyze: 1) the number of receptors contributing to three potential signalling events and six functional responses and 2) the evolution of these responses once ligand binding is interrupted. We find that the half-optimal elevations of the potential signals are produced by less than 1% occupancy (Ca2+) or 1-3% occupancy (cAMP, membrane depolarization). In contrast, actin polymerization and a rapid light scatter response are elicited by less than 0.1% occupancy. Half-optimal elastase release and degranulation require approximately 3% occupancy. While half-optimal O2- production and aggregation require approximately 30% occupancy, the half-optimal rate of O2- production requires less than 10% occupancy. To resolve the apparent lack of correlation between the responses and the signals we examined their time courses following the pulse of stimulation. At least four responses and one signal are transient and decay while occupied receptors remain on the membrane surface. These include the Quin 2-Ca2+ signal, actin polymerization, the light scatter response, O2- generation, and aggregation. Ca2+ elevation is correlated with the responses in that: 1) each of these responses is transient unless new receptors are occupied; 2) occupancy of nearly all of the receptors contributes to the time course of these responses; 3) when binding is interrupted, the responses decay with a half-time of 15 s, following a latency of approximately 10 s or less (except for disaggregation where latency is 30-40 s). We discuss evidence in support of the hypothesis that transient cell responses arise from transient receptor activation.

The activation of Hageman factor in solid and fluid phase has been analyzed. Activation of highly purified Hageman factor occurred after it interacted with and became bound to a negatively charged surface. Activation was observed in the absence of enzymes that are inhibitable with diisopropylfluorophosphate, phenyl methyl sulfonyl fluoride and ϵ-amino-n-caproic acid. The binding of [125I]Hageman factor to the negatively charged surface was markedly inhibited by plasma or purified plasma proteins. Activation of Hageman factor in solution (fluid phase) was obtained with kallikrein, plasmin, and Factor XI (plasma thromboplastin antecedent). Kallikrein was greater than 10 times more active in its ability to activate Hageman factor than plasmin and Factor XI. The data offer a plausible explanation for the finding that highly purified kallikrein promotes clotting of normal plasma. In addition, the combined results of this and previously reported data from this laboratory indicate that the reciprocal activation of Hageman factor by kallikrein in fluid phase is essential for normal rate of activation of the intrinsic-clotting, kinin-forming, and fibrinolytic systems. Activation of Hageman factor was associated with three different structural changes in the molecule: (a) Purified Hageman factor, activated on negatively charged surfaces retained its native mol wt of 80–90,000. Presumably a conformational change accompanied activation. (b) In fluid phase, activation with kallikrein and plasmin did not result in cleavage of large fragments of rabbit Hageman factor, although the activation required hydrolytic capacity of the enzymes. (c) Activation of human Hageman factor with kallikrein or plasmin was associated with cleavage of the molecule to 52,000, 40,000, and 28,000 mol wt fragments. Activation of rabbit Hageman factor with trypsin resulted in cleavage of the molecule into three fragments, each of 30,000 mol wt as noted previously. This major cleavage occurred simultaneously with activation.

The ability of the two forms of activated Hageman factor (HFa) produced during contact activation of plasma to activate prekallikrein and factor XI was studied. alpha-HFa, defined as an 80,000 mol wt two-chain enzyme which remains bound to the surface was capable of cleaving surface-bound prekallikrein and factor XI. beta-HFa, a 28,000 mol wt single chain molecule, released from the surface during contact activation was able to cleave prekallikrein but showed no activity on factor XI. Cleavage of prekallikrein by beta-HFa occurred irrespective of whether the substrate was surface-bound or in solution. Cleavage of factor XI occurred only when it was surface bound and only the alpha-form of HFa was capable of this proteolytic action. Factor XI was found to remain bound to the surface while prekallikrein and kallikrein rapidly dissociated from the surface into the supernate. These findings suggest that the initiation of intrinsic coagulation through the activation factor XI is a localized event occurring at the site of contact activation and is the result of the action of alpha-HFa. By contrast, kinin generation and fibrinolysis resulting from the formation of kallikrein can be initiated either at the site of contact activation, by alpha-HFa action, or throughout the plasma, by beta-HFa; further dissemination of these activities is assured by the rapid dissociation of kallikrein itself from the surface.

Parallel cytometric and fluorimetric analyses of the interaction of a fluoresceinated N-formyl hexapeptide (N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein, Nle = norleucine) with its receptors on human neutrophils are presented. The cytometric analyses take advantage of the ability of the fluorescence flow cytometer to discriminate free and receptor-bound ligand in a homogeneous real-time assay. The spectrofluorometric analysis relies on a high affinity antibody to fluorescein to discriminate free and bound ligand. We find that the number of receptors for formyl peptides on the surface of a resting cell is 53,000 +/- 13,000 (Kd approximately 0.6 +/- 0.2 nM). We use commercially available cytometric standards to calibrate the cytometer and we obtain similar values for the number of receptors. The temperature dependence of the kinetics of ligand-receptor interactions have been examined. The association rate constant varies from approximately 3 X 10(8) M-1 min-1 at 4 degrees C to approximately 10(9) M-1 min-1 at 37 degrees C (delta H approximately 8 kcal/mol). While ligand internalization is blocked at 4 degrees C, at 37 degrees C internalization proceeds at an initial rate of approximately 24% of the occupied receptors/min following a latency period of approximately 20-30 s. Intermediate rates and longer latency periods are found at 15 and 25 degrees C. Dissociation of the ligand is heterogeneous and depends both on the length of time of association and the temperature. After short periods of association, the ligand dissociates with t1/2 approximately 1-5 min. After longer periods (30 min at 15 degrees C or 100 min at 4 degrees C), but while the ligand-receptor complex remains on the cell surface, t1/2 increases to greater than 30 min. It appears that the ligand-receptor complex undergoes an alteration in affinity, with a time course at elevated temperatures, which parallels or lags behind the time course of the participation of the occupied receptors in cell activation.

Experiments were performed to examine how human granulocytes process the chemotactic peptide N-formyl-Met-Leu-Phe after stimulation by the same peptide. Purified human granulocytes were stimulated with 50 nM N-formyl-Met-Leu-[3H]Phe at 37 degrees C for various times, washed, lysed by N2 cavitation, and fractionated by isopycnic sucrose density gradient sedimentation. The major subcellular fractions identified were plasma membrane, Golgi, granules, endoplasmic reticulum, and mitochondria. After 1 min of stimulation, radioactivity was found only in the plasma membrane (sedimentable) and cytosol (soluble) fraction. At 5, 10, and 25 min, radioactivity also appeared in a sedimentable, low density fraction (25-28% sucrose) enriched in galactosyl transferase activity and containing Golgi structures. The accumulation in the sedimentable fractions was maximal after 5 min but continued to increase linearly in the cytosol fraction. Incorporation of radioactivity into cells or membrane and soluble fractions was 60 to 85% specific and was inhibited if incubation with N-formyl-Met-Leu-[3H]Phe was performed at 4 degrees C. 80-90% of the radiolabel in the plasma membrane or Golgi-containing fractions remained sedimentable despite freeze thawing or sonication. Solubilization of these fractions in Triton X-100 followed by Sepharose 4B column chromatography revealed that the radiolabel eluted in the void volume. Our results are consistent with internalization which proceeds by passage of an occupied receptor in a high affinity, supramolecular complex from the plasma membrane to the Golgi followed by accumulation of peptide in the cytosol.

H2O2, in concentrations achieved in the proximity of stimulated leukocytes, induces injury and lysis of target cells. This may be an important aspect of inflammatory injury of tissues. Cell lysis in two target cells, the murine macrophage-like tumor cell line P388D1 and human peripheral lymphocytes, was found to be associated with activation of poly(ADP-ribose) polymerase (EC 2.4.2.30), a nuclear enzyme. This enzyme is activated under various conditions of DNA damage. Poly(ADP-ribose) polymerase utilizes nicotinamide adenine dinucleotide (NAD) as substrate and has been previously shown to consume NAD during exposure of cells to oxidants that was associated with inhibition of glycolysis, a decrease in cellular ATP, and cell death. In the current studies, inhibition of poly(ADP-ribose) polymerase by 3-aminobenzamide, nicotinamide, or theophylline in cells exposed to lethal concentrations of H2O2 prevented the sequence of events that eventually led to cell lysis--i.e., the decrease in NAD, followed by depletion of ATP, influx of extracellular Ca2+, actin polymerization and, finally, cell death. DNA damage, the initial stimulus for poly(ADP-ribose) polymerase activation, occurred despite the inhibition of this enzyme. Cells exposed to oxidant in the presence of the poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide failed to demonstrate repair of DNA strand breaks.

During protein depletion produced by plasmapheresis and a very low protein diet there is a proportionately greater decrease in extravascular, extracellular fluid protein than in plasma protein. A shift in the normal ratio of protein in these 2 compartments, approximately 1 to 1 in the dog, to over 2 to 1 as a result of depletion indicates an important, labile source of reserve protein for the plasma in the interstitial fluids. This reserve source is limited since a maximum drop of 50 per cent in the total exchangeable pool and of 75 per cent in the extravascular, extracellular protein occurred after both shorter and longer periods of depletion. Under the rigid conditions of these experiments additional plasma protein removal was associated with loss of weight despite adequate caloric intake. Investigation of the status of the interstitial fluid proteins in other conditions associated with disturbed protein metabolism seems warranted.

Two mechanisms have been described whereby rabbit platelets in vitro may be induced to release their contained histamine by the reaction of antigen and antibody. Both processes require the participation of the complement system. In the first, the adherence of platelets to particulate antigens such as zymosan or erythrocytes which have fixed complement through the third component was followed by histamine release. Plasma lacking C6 activity was fully active in this system. In the second mechanism, the reaction of soluble antigen with antibody in the presence of plasma also caused release of histamine from platelets and platelet clumping was observed. The release process, which appeared to follow the adherence of platelets to the immune complex, required the action of C6 and perhaps the later-acting components. No evidence could be obtained that a soluble factor was produced which caused the release. Instead, an inhibitor of the release process was detected after incubation of antigen, antibody, and plasma. Antibody preparations capable of giving complement-dependent PCA reactions in rabbits were also shown to induce the release of histamine from platelets in vitro.