The effects of basic substances and acidic ionophores on the digestion of exogenous and endogenous proteins in mouse peritoneal macrophages.

Basic substances and acidic ionophores that increase the lysosomal pH in cultured macrophages (Ohkuma, S., and B. Poole, 1978, Proc. Natl. Acad. Sci. USA., 75:3327-3331; Poole, B., and S. Ohkuma, 1981, J. Cell Biol., 90:665-669) inhibited the digestion of heat-denatured acetylated bovine serum albumin (BSA) taken up by the cells. For several substances, the shift in pH sufficed to explain the inhibition of proteolysis. Additional effects, presumably on enzyme activities, have to be postulated for tributylamine, amantadine, and chloroquine. Sodium fluoride (10 mM) had no significant effect on the breakdown of BSA by macrophages. The breakdown of endogenous macrophage proteins, whether short lived or long lived, was inhibited approximately 40% by 10 mM NaF and 30%, or sometimes less in the case of long-lived proteins, by 100 microM chloroquine. When the cells were supplied with BSA, a mixture of cell proteins, or even inert endocytosible materials, the breakdown of endogenous long-lived proteins and the inhibitory effect of chloroquine on this process were selectively reduced. Inhibition of endocytosis by cytochalasins B or D did not affect the chloroquine-sensitive breakdown of endogenous proteins, indicating that the proteins degraded by this process were truly endogenous and not taken in from the outside by cellular cannibalism. On the other hand, when macrophage proteins were supplied extracellularly, their breakdown occurred at the same rate for short-lived and long-lived proteins, and it was strongly inhibited by chloroquine and not by NaF. It is concluded from these results that the breakdown of endogenous proteins, both short-lived and long-lived, probably takes place partly (approximately 30%) in lysosomes and partly through one or more nonlysosomal mechanism(s) unaffected by chloroquine and presumably susceptible to inhibition by fluoride. A difference must exist between short-lived and long-lived proteins in the manner in which they reach lysosomes or are handled by these organelles; this difference would account for the selective effect of the supply of endocytosible materials on the lysosomal processing of long-lived proteins.

Metabolic fate of liposomal phosphatidylinositol in murine tumor cells: implications for the mechanism of tumor cell cytotoxicity.

The mechanism of the previously reported cytotoxicity of liposomes containing plant phosphatidylinositol (PI) against numerous tumor cell lines was examined in detail by using liposomes containing synthetic PI specifically labeled either with radioactive myo-inositol, or in the sn-2 position with radioactive linoleic acid, oleic acid, or arachidonic acid. The uptake of liposomal PI by N4TG1 neuroblastoma cells increased with time and was dependent on the nature of the fatty acids. Uptake was highest with liposomal PI containing linoleic acid followed by arachidonic acid and then by oleic acid. The cellular fate of liposomal PI was determined by analysis of radioactive metabolites present in extracts of tumor cell lipids. Appearance of liposomal PI metabolic products in the tumor cells was correlated with thymidine uptake as a measure of viability. After 3 h incubation of cells with PI liposomes it was found that the release of both radioactive liposomal fatty acids (and probably also lyso-Pl) and radioactive diglycerides was correlated inversely with the cellular uptake of [methyl-3H]thymidine and uptake of [3H]myoinositol. An experiment in which liposomes were prepared both from animal Pl which contained predominantly saturated fatty acids in the sn-2 position and an increasing mole fraction of a synthetic Pl containing radioactive linoleic acid in the sn-2 position established that the amount of Pl containing linoleic acid in the sn-2 position could be correlated with a decrease in the amount of thymidine uptake by tumor cells. The above results clearly established that phospholipases A2 and C in the tumor cells were responsible for the formation of metabolites of liposomal Pl, and these metabolic products might have been responsible for cytotoxicity and cell death.

Effects of selective serotonin reuptake inhibitors on platelet serotonin parameters in major depressive disorder.

The effects of treatment with serotonin (5-HT) reuptake inhibitors on platelet 5-HT2 receptors, 5-HT reuptake sites an 5-HT uptake were studied in a double-blind trial comparing two selective serotonin reuptake inhibitors (SSRI), paroxetine, and fluoxetine, for the treatment of major depression. Hamilton Depression Rating Scale (HAM-D) scores and platelet 5-HT parameters were determined in 21 depressed patients at baseline, after 4 and 8 weeks of treatment, and were compared to 21 healthy controls. Antidepressant treatment did not significantly alter the density of 5-HT reuptake sites, labelled with [3H]paroxetine, or 5-HT2 receptors, labelled with [3H]LSD. However, a strong correlation was observed between the HAM-D suicidality item and 5-HT2 receptor density at baseline. A marked increase in platelet 5-HT2 receptors at baseline was observed in suicidal depressed patients compared to those with no suicidal ideation and healthy controls. Changes in [3H]paroxetine Bmax and in [3H]5-HT uptake significantly correlated with change in HAM-D score at 4 and 8 weeks respectively. These results confirm previous reports of an association between suicidality and platelet 5-HT2 receptor upregulation. Our data also lends support to the use of platelet 5-HT parameters as indicators of antidepressant efficacy, particularly in suicidal depressed patients.

Platelet serotonin measures in primary dysthymia.

Serotonergic function in 22 patients with primary dysthymia and 22 normal volunteers was evaluated by measuring [3H]serotonin uptake and [3H]paroxetine binding in platelets. A significantly lower maximum rate of serotonin uptake was noted in the dysthymic patients than in the normal subjects, indicating a possible serotonergic dysfunction in dysthymia. However, the values for parameters of paroxetine binding were similar in the two groups.

Synthesis and characterization of an aryl-azidoparoxetine. A novel photo-affinity probe for serotonin-transporter.

Paroxetine is an effective antidepressant drug and potent serotonin (5-HT) uptake inhibitor. It selectively labels 5-HT transporter on platelets and neurons. We report here the synthesis of an aryl-azido derivative of paroxetine, which is a novel photoactive and irreversible ligand for the [3H]paroxetine binding site on the platelet 5-HT transporter. The compound inhibited [3H]paroxetine binding (IC50, 55 nM) and 5-HT uptake (IC50, 12 nM) at equilibrium conditions and inactivated 10-20% of [3H]paroxetine binding sites upon irradiation at 320 nm. SDS-PAGE of platelet protein extract labelled with the radioactive analogue of the synthesized probe revealed the presence of four radioactive bands of which the 71-kDa one was the most prominent.

Interaction of lectins with human platelet serotonin transporter.

Lectin affinity chromatography was used to demonstrate the glycoprotein nature of the serotonin (5-HT) transporter. The human platelet transporter protein was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), labelled with [3H]cyanoimipramine and chromatographed on lectin columns. Wheat germ, gorse seed and lentil lectin-agarose columns specifically retained 5-HT transporter. Peanut and horse gram lectins were ineffective. Concanavalin A showed high non-specific adsorption. Binding of [3H]imipramine to platelet membranes or solubilized 5-HT transporter was not affected by lectins. These data suggest that the lectin interaction with 5-HT transporter is extrinsic to the antidepressant binding site.

Inhibition of platelet [3H]-imipramine binding by human plasma protein fractions.

Inhibition of high-affinity [3H]-imipramine binding to platelet membranes by human plasma fractions and isolated plasma proteins was investigated. Several plasma proteins were found to contribute to the observed apparent inhibition and this contribution was assessed in terms of inhibitor units. Alpha 1 acid glycoprotein, high density and low density lipoprotein, IgG and alpha 1-antitrypsin were identified as effective non-specific inhibitors. Alpha-1-acid glycoprotein was confirmed to be the most potent plasma protein inhibitor. Cohn fractions were evaluated for the presence of the postulated endocoid of [3H]-imipramine binding site.

Further evidence for the existence of endogenous serotonin uptake inhibitors and their purification by calmodulin affinity chromatography.

Experimental evidence suggests the occurrence of endogenous antidepressant-like compounds in the brain, blood plasma, and urine. After extensive purification by calmodulin-sepharose affinity chromatography, and further purification of the urine-derived sample by exclusion chromatography, at least three distinctive fractions have been identified. These fractions effectively inhibited serotonin uptake, imipramine, and/or paroxetine binding, and they apparently contained some compounds that were recognized by the anti-imipramine or anti-paroxetine antibodies. Their identification may have significant implications for studies of affective illness.

Anti-imipramine antibodies recognize endogenous serotonin uptake and imipramine binding inhibitors.

Calf brain and human platelet extracts purified by Bio-Gel P2 column chromatography contained substances that inhibited serotonin uptake and 3H-imipramine binding. Some of these endogenous substances were also recognized by rabbit antibodies produced against imipramine. The data suggest the possible existence of endogenous serotonin uptake modulators, which may possess a partial molecular structure similar to that identified by the antibodies.

Platelet serotonergic indices in major depression: up-regulation of 5-HT2A receptors unchanged by antidepressant treatment.

This study examined, in the largest sample of major depressives reported so far, platelet serotonergic parameters (5-HT uptake, [3H]paroxetine binding and 5-HT2A receptors measured by [3H]LSD binding) in 60 antidepressant-free depressed patients and 40 age- and gender-matched control subjects before treatment, and in 45 major depression patients during treatment with antidepressants. We found that, at baseline, the density (Bmax) of 5-HT2A receptors was significantly higher (by 39%) in depressed patients than in controls. Suicidal patients had significantly higher Bmax values than controls or non-suicidal patients. The rate of serotonin uptake (Vmax), but not the uptake at a single concentration, was significantly higher in depressed patients, particularly in females. There was no significant difference between the Kd or Bmax of [3H]paroxetine binding in control and depressed subjects. Treatment with antidepressant drugs of different pharmacological profile had no significant effect on the density of 5-HT2A receptors, nor did the receptor number predict the response to treatment. The affinity of serotonin uptake site for 5-HT and [3H]paroxetine significantly decreased during treatment with antidepressants, particularly SSRIs. Suppression of 5-HT uptake correlated with decreases in Hamilton depression (HAMD) scores. Our data suggest that the increased density of platelet 5-HT2A receptors may be associated with untreated major depression in antidepressant-free depressed patients, in particular those with suicidal thoughts. The persistence after antidepressant treatment and clinical improvement would suggest that up-regulation of 5-HT2A receptors is a trait rather than state phenomenon. Correlation of 5-HT uptake suppression with decreases in HAMD scores suggests that serotonin uptake inhibition is a relevant factor in antidepressant drug effect and clinical improvement.

Co-encapsulation of cyclosporin and insulin by liposomes.

Evidence for an association of both hydrophilic insulin and hydrophobic cyclosporin with liposomes prepared from egg yolk lecithin, cholesterol, and stearylamine (7:2:2.25 molar ratio) was obtained by Sepharose-4B gel filtration. The method used to prepare unilamellar liposomes containing 29.7 nmol cyclosporin and 2.3 nmol insulin per mu mol of liposomal lecithin is described.

Severe haemorrhagic diathesis in an adult patient with cystic fibrosis after long-term antibiotic treatment of pulmonary infection.

We describe the case of a 22 yr old male patient with cystic fibrosis, who, after long-term antibiotic treatment of pulmonary infection, developed a haemorrhagic diathesis with severe bleeding from the mucus membrane of the mouth, and haematuria. Rapid recovery was observed after infusion of vitamin K. During 8 months of follow-up, no evidence of recurrence of the clotting disturbances and anaemia were noted. The combination of impaired absorption of vitamin K due to underlying disease with the antibiotic-induced suppression of vitamin K synthesis by intestinal bacteria could be a possible explanation for this disorder.

Phosphatidylinositol movement between isolated microsomal and mitochondrial membranes.

Transfer of membrane-bound phosphatidyl-[2'-3H]inositol from microsomal to unlabelled mitochondrial and from mitochondrial to unlabelled microsomal membranes was studied using partially purified cytosol proteins isolated from guinea pig liver cytosol. In the absence and presence of these proteins the amounts of phosphatidylinositol transfer from microsomal to mitochondrial membranes were approximately 21 and 33%, respectively, and the amounts from mitochondrial to microsomal membranes were approximately 31 and 39%, respectively. The release of phosphatidyl-[2'-3H]inositol from microsomal membranes in the absence of mitochondria was dependent on concentration of cytosol proteins. Two mechanisms for movement between membranes are proposed. In cytosol-protein-independent movement of phosphatidyl-[2'-3H]inositol from microsomal to mitochondrial membranes, a direct contact between membranes is required, since phosphatidyl-[2'-3H]inositol was not detected in the reaction medium. In the cytosol-protein-catalyzed transfer, formation of phosphatidyl-[2'-3H]inositol - cytosol protein complex is postulated, since phosphatidyl-[2'-3H]inositol was released into the reaction medium and its movement proceeded from mitochondrial to microsomal membranes in the presence of partially purified cytosol proteins. Thus, contact between the two membranes is probably not necessary for this transfer. Implications for the movement of phospholipids between biological membranes are discussed.

Isolation and some molecular parameters of elastase-like normal proteinases from horse blood leucocytes.

Cytoplasmic granules were isolated from horse blood polymorphonuclear leucocytes by the heparin method and extracted with 0.9% NaCl by repeated freezing. Soluble proteins were separated on a column of Sephadex G-75 followed by chromatography on a column of CM-Sephadex with a NaCl gradient. Gel filtration, density-gradient centrifugation, isoelectric focusing and 0.1% sodium dodecyl sulphate/polyacrylamide-gel electrophoresis at pH 7.0 and at pH 4.5 were used to determine molecular parameters of proteinases. Three enzymes hydrolysing both casein and N-benzyloxycarbonyl-L-alanine nitrophenyl ester were found in the granule extract: proteinase 1, mol.wt. 38000, pI5.3; proteinase 2A, mol.wt. 24500, pI8.8; and proteinase 2B, mol.wt. 20500, pI above 10. The latter two elastase-like proteinases were purified to apparent homogeneity.