Relationship between the intracellular cyclic adenosine 3':5'-monophosphate level of tumor cells and their sensitivity to killing by antibody and complement.

Metabolic inhibitors which have been shown to increase the sensitivity of the guinea pig hepatoma, line 10, to antibody-guinea pig complement killing were tested for their effect on the intracellular cyclic adenosine 3':5'-monophosphate (cAMP) level of the line 10 cells. It was found that cells rendered sensitive to antibody-guinea pig complement killing following drug treatment showed a decrease in their intracellular cAMP levels. Increased susceptibility to lysis was always associated with decreased intracellular cAMP levels. Optimal doses of certain inhibitors effectively increased the sensitivity of the cells to complement-dependent lysis and decreased the intracellular cAMP levels. Other drugs which did not increase sensitivity of the cells to lysis also failed to decrease the intracellular cAMP levels. No experimental conditions were found in which intracellular cAMP levels were decreased without an increase in the susceptibility of the cells to antibody-guinea pig complement killing.

The interaction of alpha-1-antitrypsin with soluble and sepharose-bound elastase.

The products resulting from the interaction of alpha-1-antitrypsin with elastase were examined with polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate, and by affinity chromatography. Five products of the reaction can be identified by polyacrylamide disc gel electrophoresis. Two products are complexes between alpha-1-antitrypsin and elastase (73 800 and 58 300 daltons). Two additional products are identical to fragments of alpha-1-antitrypsin which can be washed from a column of Sepharose-bound elastase immediately after alpha-1-antitrypsin is applied to the column. The larger component about 50 000 daltons, reacts with antiserum to alpha-1-antitrypsin, and does not inhibit enzymes. Together, these two products have an amino acid analysis similar to alpha-1-antitrypsin. These two fragments are probably hydrolytic products of the interaction of elastase with alpha-1-antitrypsin which is biologically inactive. The fifth product is probably a fragment of alpha-1-antitrypsin missing from the low molecular weight complex. The components of the complexes can be separated from each other by a mild nucleophilic attack. Small quantities of alpha-1-antitrypsin can be displaced from the elastase affinity column by phenyl methane sulfonyl fluoride. In conclusion, porcine pancreatic elastase forms two complexes with alpha-1-antitrypsin. One or both complexes can be split by alkali.

Cardiotoxin from cobra venom increases the level of phosphatidylinositol 4-monophosphate and phosphatidylinositol kinase activity in two cell lines.

In rat basophilic leukemia-2H3 (RBL-2H3) and Madin-Darby canine kidney (MDCK) cells, cardiotoxin from cobra venom induced a marked decrease in the level of [3H] phosphatidylinositol and a corresponding increase in the level of [3H]phosphatidylinositol 4-monophosphate over the course of 20 min as demonstrated in cells that had been labeled to equilibrium with [3H]inositol. The effect was dependent on the concentration (5-30 micrograms/ml) of the toxin. In plasma membrane-enriched fractions isolated from the two cell lines, the cardiotoxin enhanced the endogenous activity of phosphatidylinositol kinase especially at temperatures above 14 degrees C. In RBL-2H3 cells, cardiotoxin also induced release of substantial amounts of histamine and lactate dehydrogenase. The release of histamine, but not of lactate dehydrogenase, was totally dependent on external calcium and this release probably represented an exocytotic response of the cells to cardiotoxin. Although, initially, treatment with the toxin did not impair antigen-induced hydrolysis of inositol phospholipids or prevent the antigen-induced rise in the concentration of cytosol Ca2+, prolonged exposure to the toxin did result in a progressive loss of responsiveness of RBL-2H3 cells to antigen.

Triiodothyronine augments the number of membrane-bound (Na+-K+)-adenosine triphosphatase units, but does not affect the sedimentation properties of plasma membrane components.

We have previously demonstrated that T3 enhanced the de novo synthesis of renal cortical (Na+-K+)-dependent ATPase (NaK-ATPase) in the rat. A purified membrane fraction obtained from successive centrifugation of renal cortical crude homogenate was used in the above studies. To rule out a possible effect of T3 on plasma membrane properties, such as the sedimentation characteristic of NaK-ATPase, we have presently observed T3-dependent increases in the activity and number of NaK-ATPase units in a crude homogenate of rat renal cortex. The following results were obtained which substantiate a specific effect of T3 on the membrane-bound NaK-ATPase system. 1) Compared to the hypothyroid state, 43% and 44% increases in the activity and number of NaK-ATPase units, respectively, were observed in crude renal cortical homogenate of T3-treated hypothyroid rats. 2) In comparison with the crude homogenate prepared from hypothyroid rats, 4.8- and 113-fold increases in the specific activity of NaK-ATPase were obtained in the L and J fractions, respectively. Increases of similar relative magnitude in the L and J fractions were also shown in T3-treated hypothyroid and euthyroid rats. 3) No difference in the recovery of the number of NaK-ATPase units was observed from successive steps of the purification under different thyroid states. 4) Treatment of renal cortices from hypothyroid, T3-treated hypothyroid, and euthyroid rats with deoxycholate increased to the same extent NaK-ATPase activity and phosphorylated intermediate formation.

Carrageenan-stimulated release of arachidonic acid and of lactate dehydrogenase from rat pleural cells.

Cells isolated from the rat pleural cavity consist mainly of macrophages, mast cells, eosinophils, and lymphocytes. Isolated pleural cells labeled with [14C]arachidonic acid released appreciable amounts (approximately 12%) of radiolabel upon exposure to pharmacological concentrations of carrageenan (1-100 micrograms/ml). The release of radiolabel was decreased by an inhibitor of phospholipase A2 (p-bromophenacyl bromide) but not by an inhibitor of arachidonate cyclooxygenase (indomethacin). The released products were arachidonic acid and, to a much lesser extent, prostaglandin E2 and leukotriene C4. The release of radiolabel was associated with release of cytosolic lactate dehydrogenase over the same range of carrageenan concentrations. Time-course studies indicated that release of radiolabel preceded that of lactate dehydrogenase. Since p-bromophenacyl bromide blocked stimulated release of radiolabel but did not prevent release of lactate dehydrogenase, it is unlikely that increase in arachidonate causes carrageenan-induced cell damage. Nevertheless, the question of whether the activation of phospholipase A2 in the pleural cells, most probably the macrophages, was sufficient to initiate the carrageenan-induced inflammatory response requires further study. Cytotoxicity which was apparent with as little as 5 micrograms/ml of carrageenan, may have been a significant consequence of carrageenan action.

Inflammatory response induced by intrapleural injection of antiserum to IgE in rat. An evaluation of the role of histamine.

Intrapleural injection of antiserum to rat IgE (anti-IgE) into rats resulted in release of histamine from mast cells and rapid effusion of fluid and plasma proteins into the pleural cavity. By 4 hr this was followed by infiltration of neutrophils. These responses were dependent on the amount of anti-IgE injected, and maximal responses were greater than those obtained with compound 48/80. The effusion of fluid and protein, but not the infiltration of cells, was partially suppressed by prior treatment with the H1 histamine receptor antagonist mepyramine (5 mg/kg, s.c.) or the H2 antagonist metiamide (100 mg/kg, s.c.) and was almost totally suppressed (85-88%) when both drugs were administered simultaneously. Neither methysergide (1 and 4 mg/kg, s.c.) nor indomethacin (5 and 10 mg/kg, i.v.) had an effect on the responses to anti-IgE. Although it seemed likely that histamine was a primary mediator of increased vascular permeability, the intrapleural injection of histamine agonists or histamine in large amounts (50 micrograms) provoked a much less intense response than did anti-IgE. The effects of injected histamine may not, therefore, mimic those induced by histamine released from mast cells in situ. The intrapleural injection of histamine releasers such as anti-IgE may serve as a useful model to test the therapeutic efficacy of antihistamine drugs. The present results also confirm previous reports that localized neutrophil infiltration occurs after mast cell degranulation.

Effect of indomethacin on generation of chemotactic activity in inflammatory exudates induced by carrageenan.

Chemotactic activity of protein origin was demonstrated in carrageenan-induced pleural exudates by the chemotactic response of neutrophils in the modified Boyden chamber. The activity was partly neutralized by monospecific antisera to complement component 5 and was destroyed by trypsin and chymotrypsin treatment but it differed from that in rat serum or plasma in that it was stable for 30 min at 56 degrees C. Indomethacin (5 mg/kg i.v.) reduced equally protein content (56%) and total chemotactic activity (58%); i.e., chemotactic activity/mg of exudate protein was unchanged. Intrapleural injection of autologous or homologous serum also induced an infiltration of neutrophils; the protein content of the pleural fluid decreased by 60-70% in 4 h, whereas with carrageenan there was a progressive increase in exudate protein. When serum was injected in two doses to maintain protein levels comparable to those found following carrageenan injection, the number of neutrophils in the exudates was also comparable. In contrast to carrageenan, the response to serum was not inhibited by indomethacin. From these and other data we suggest that the exudate chemotactic activity is generated from plasma protein and that indomethacin acts primarily to reduce extravasation of plasma and consequently generation of chemotactic activity.

Fabrication of high-aspect-ratio Fresnel zone plates by e-beam lithography and electroplating.

The fabrication of gold Fresnel zone plates, by a combination of e-beam lithography and electrodeposition, with a 30 nm outermost zone width and a 450 nm-thick structure is described. The e-beam lithography process was implemented with a careful evaluation of applied dosage, tests of different bake-out temperatures and durations for the photoresist, and the use of a developer without methylisobutylketone. Electrodeposition with a pulsed current mode and with a specially designed apparatus produced the desired high-aspect-ratio nanostructures. The fabricated zone plates were examined by electron microscopy and their performances were assessed using a transmission X-ray microscope. The results specifically demonstrated an image resolution of 40 nm.

Time course of the renal response to triiodothyronine in the rat.

Experiments were carried out to compare temporal changes in glomerular filtration rate (GFR), filtered Na+ load, and renal cortical (Na+ + K+)-adenosine triphosphatase (Na-K-ATPase) activity in the hypothyroid rat after administration of a single dose of triiodothyronine (T3) (50 microgram/100 g body wt). The cortex showed an increase in Na-K-ATPase at 24 h and progressive increases to a peak of 62% at 48 h. GFR and filtered Na+ load showed no changes at 24 and 48 h. At 72h, however, significant increases of 62 and 63% (per rat) were observed in GFR and filtered Na+ load, respectively. The results show that the early increase in Na-K-ATPase activity upon T3 treatment precedes the increases in GFR and filtered Na+ load, suggesting a direct effect of T3 on the regulation of Na-K-ATPase activity in the hypothyroid rat kidney cortex, rather than a secondary response to a primary increase in filtered Na+ load as proposed previously.

Anti-inflammatory drugs alter amino acid transport in HTC cells.

Following the addition of indomethacin to exponentially growing rat hepatoma cell (HTC) cultures, cells accumulated in the G1 phase. Over the course of several hours, specific changes in membrane transport accompanied this inhibition. Indomethacin stimulated the uptake of 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (BCH), inhibited the Na+-dependent active transport of alpha-aminoisobutyric acid (AIB), and methylaminoisobutyric acid (MeAIB), but had no effect on the uptake of thymidine, uridine, or 2-deoxyglucose. The stimulation of BCH transport was immediate and reversible. The uptake of BCH was Na+-independent and only slightly depressed by metabolic inhibitors (10 to 20%). Inhibition of AIB and MeAIB uptake, which was of gradual onset, and was observed with several anti-inflammatory drugs and was dependent on the concentration of drugs. Upon removal of drug, AIB and MeAIB transport gradually increased and reached a maximum prior to resumption of DNA synthesis and cell division. Thee effects involved changes in Vmax only. Neither the apparent affinity (Km) for amino acids nor rate of exodus (k, 0.7h-1 for BCH, 1.2h-1 for AIB) were altered. The uptake of MeAIB (and AIB) occurred by a low and a high affinity (Km = 0.27 mM) component. Indomethacin inhibited specifically the high affinity component which was shown to be Na+- and energy-dependent. Although this component was inhibited by treatment with agents that lowered ATP levels or blocked protein synthesis, the anti-inflammatory drugs appeared not to act through these mechanisms.