V-ATPase regulates communication between microvascular endothelial cells and metastatic cells.

To metastasize distant organs, tumor cells and endothelial cells lining the blood vessels must crosstalk. The nature of this communication that allows metastatic cells to intravasate and travel through the circulation and to extravasate to colonize different organs is poorly understood. In this study, we evaluated one of the first steps in this process­the proximity and physical interaction of endothelial and metastatic cells. To do this, we developed a cell separator chamber that allows endothelial and metastatic cells to grow side by side. We have shown in our previous studies that V-ATPases at the cell surface (pmV-ATPase) are involved in angiogenesis and metastasis. Therefore, we hypothesized that the physical proximity/interaction between endothelial and metastatic cells expressing pmV-ATPase will increase its activity in both cell types, and such activity in turn will increase pmV-ATPase expression on the membranes of both cell types. To determine pmV-ATPase activity we measured the proton fluxes (JH+) across the cell membrane. Our data indicated that interaction between endothelial and metastatic cells elicited a significant increase of JH+ via pmV-ATPase in both cell types. Bafilomycin, a V-ATPase inhibitor, significantly decrease JH+. In contrast, JH+ of the non-metastatic cells were not affected by the endothelial cells and vice-versa. Altogether, our data reveal that one of the early consequences of endothelial and metastatic cell interaction is an increase in pmV-ATPase that helps to acidify the extracellular medium and favors protease activity. These data emphasize the significance of the acidic tumor microenvironment enhancing a metastatic and invasive phenotype.

Clinical concentrations of doxorubicin inhibit activity of myocardial membrane-associated, calcium-independent phospholipase A(2).

Use of the anticancer antibiotic doxorubicin continues to be limited by its cumulative dose-related cardiotoxicity. Our study reports inhibition of myocardial intracellular calcium-independent phospholipase A(2) (iPLA(2)) activity by clinically relevant concentrations of the drug. The effect was first shown in vitro using suspensions of freshly isolated rat and rabbit cardiomyocytes. Addition of 0.1-10 microM doxorubicin to these cells led to a concentration- and time-dependent inhibition of total iPLA(2), as measured using (16:0, [(3)H]18:1) plasmenylcholine and phosphatidylcholine substrates in the presence or absence of calcium. Subcellular fractionation into cytosolic and membrane fraction revealed that the drug selectively inhibits membrane-associated iPLA(2) activity, without altering activity of the cytosolic enzyme. Doxorubicin treatment of cells prelabeled with [H(3)]arachidonic acid led to a depression of baseline arachidonic acid release levels, corroborating iPLA(2) inhibition. Reducing agents blocked PLA(2) inhibition in cardiomyocyte suspensions, suggesting that the doxorubicin effect is mediated by oxidation of susceptible cysteines. In vivo experiments, in which adults rats were i.v. injected with a bolus dose of 4 mg/kg doxorubicin, confirmed in vitro findings, revealing a 2-fold decrease in membrane-associated Ca(2+)-independent iPLA(2) activity in the heart tissue of treated animals. The observed phenomenon has important implications for myocyte signaling cascades and membrane remodeling.

Localized injury in cardiomyocyte network: a new experimental model of ischemia-reperfusion arrhythmias.

We developed a new experimental approach to study the effects of local injury in a multicellular preparation and tested the ability of the method to induce reperfusion arrhythmias in cardiomyocyte monolayers. A small region of injury was created using geometrically defined flows of control and ischemia-like solutions. Calcium transients were acquired simultaneously from injured, control, and border zone cells using fluo 4. Superfusion with the injury solution rapidly diminished the amplitude of calcium transients within the injury zone, followed by cessation of cell beating. Reperfusion caused an immediate tachyarrhythmic response in approximately 17% of experiments, with a wave front propagating from a single cell or small cell cluster within the former injury zone. Inclusion of a gap junction uncoupler (1 mM heptanol) in the injury solution narrowed the functional border and sharply increased the number of ectopic foci and the incidence of reperfusion arrhythmias. The model holds a potential to reveal both micro- and macroscopic features of propagation, conduction, and cell coupling in the normal and diseased myocardium and to serve as a new tool to test antiarrhythmic protocols in vitro.

Cystatins from bovine brain: purification, some properties, and action on substance P degrading activity.

Two cystatins were purified from tissue extract of bovine brain by alkaline treatment, acetone fractionation, gel chromatography on Sephadex G-75, and affinity chromatography on S-carboxymethyl-papain-Sepharose. One of the inhibitors had a relatively high molecular mass, 25 kDa (HMM-cystatin) with pI 4.7, and the other, 11 kDa (LMM-cystatin) with pI 5.23. Both inhibitors showed considerable stability at pH 2 and 80 degrees C. The cystatins inhibited papain, ficin, and cathepsins B and H, but not trypsin, chymotrypsin, thermolysin, nagarse, and cathepsin D. Ki values for the complexes of papain and the inhibitors were estimated to be 2.8 x 10(-10) M for HMM-cystatin and 1.3 x 10(-9) M for LMM-cystatin. Both purified cystatins prevented degradation of substance P by soluble fraction and lysosomal extract obtained from synaptosomes, but did not suppress the cleavage of the peptide by synaptosomal plasma membranes.

Pyridoxal-5'-phosphate derivatives of substance P: preparation, spasmogenic activity, and degradation by human plasma.

Two fluorescent derivatives of substance P (SP) (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2) were prepared by chemical modification of the native peptide by pyridoxal-5'-phosphate (pyridoxal-P). The formation of both pyridoxal-P-derivatives of SP is the result of one modification procedure. The determination of the amino acid composition showed that in one of the derivatives the epsilon-amino group of the Lys residue [epsilon-(P-pxy)-SP] and in the other the epsilon-amino group of the Lys residue and also the N-terminal amino group [alpha, epsilon-di-(P-pxy)-SP] of SP had been substituted by pyridoxal-P. epsilon-(P-pxy)-SP and alpha, epsilon-di-(P-pxy)-SP have spasmogenic activity with ED50 of 1.8 X 10(-9) and 4 X 10(-9) M, respectively, tested on isolated guinea pig ileum. The fluorescence of P-pxy residues permits detection of as little as 1 pmol/ml of epsilon-(P-pxy)-SP and 0.5 pmol/ml of alpha, epsilon-di-(P-pxy)-SP. Both analogues of SP obtained are degraded by human plasma more slowly than the native peptide.

Synaptosomal degradation of substance P and some other neuropeptides.

Synaptosomes purified from spinal cord and from different rat brain areas exhibit peptide hydrolase activity, cleaving substance P (SP), bradykinin, THRH, LHRH, and neurotensin. The lowest activity for all the peptides tested was found in spinal cord, while the region with the highest degrading activity depended on the substrate: for substance P, it was striatum and cortex; for bradykinin, hypothalamus, and medulla oblongata; for THRH, striatum; for LHRH, midbrain; and for neurotensin, hippocampus. Degradation of substance P takes place at the plasma membrane of synaptosomes. Synaptosome ghosts cleave substance P (pH optimum 7-9, Km-2.5 X 10(-5) M, Vmax-130 nmol . hr-1 . mg protein-1) and also a number of its C-terminal fragments. Effects of the inhibitors show that several different classes of peptidases and proteases are involved in the degradation process. Peptide cleavage represents the probable pathway of synaptosomal inactivation of substance P.

Partial purification and some properties of a bovine brain trypsin inhibitor.

Using hemoglobin modified by pyridoxal 5'-phosphate as substrate, a trypsin inhibitor from bovine brain was purified by extraction at pH 4.5, ion-exchange chromatography on DEAE-Sephadex A-50, gel filtration on Sephadex G-100 and isoelectric focusing. On a column of Sephadex G-100 the inhibitor exhibited a molecular mass of 78 kDa. The iso-electric point of the inhibitor was 4.3-4.4. The dissociation constant (Ki) for the complex of bovine trypsin and brain inhibitor was estimated to be 3.7 X 10(-10)M as tested with a protein substrate, and 2.4 X 10(-10)M when tested with a synthetic substrate. During purification two other brain trypsin inhibitors were detected.

Hypothalamic cathepsin D: assay and isoenzyme composition.

A sensitive and convenient method of endopeptidase assay using as substrate globin modified with pyridoxal-5-phosphate was used for determination of acid proteinases in bovine hypothalamus separated by isoelectric focusing. The soluble protein fraction of hypothalamus upon elution from Sephadex gave five peaks of proteinase activity at pH 3.2. The properties indicate that these peaks of endopeptidase activity are isoenzyme forms of cathepsin D.

Acid proteinase of hypothalamus. Purification, some properties, and action on somatostatin and substance P.

In a continuing study of the physiological role of protein breakdown in the hypothalamus, acid proteinase from bovine hypothalamus was purified about 1000-fold. The molecular weight of the enzyme was approximately 50,000. Masimal activity against hemoglobin was obtained at pH 3.2-3.5; serum albumin was split much more slowly. Hypothalamus acid proteinase was partially inhibited by beta-phenyl pyruvate, or benzethonium Cl, and was completely inhibited by low concentrations of pepstatin. This proteinase splits somatostatin, substance P, and analogs of substance P. The probable sites of enzyme action on these peptides were determined by the end group dansyl technique. The enzyme, most likely cathepsin D, may play an important role in the formation and breakdown of peptide hormones in the hypothalamus.