Effect of methylguanidine and guanidine succinic acid on methemoglobin reductase activity in human red cells]

Activity of methemoglobin reductase was studied in human red cells treated with methylguanidine and guanidinosuccinic acid in concentrations similar to those in plasma of patients with chronic renal failure. Enzyme activity was measured with Richterich technique following an incubation at 37 degrees C for three hours. Results have shown that methylguanidine in concentration of 5.4 x 10(-5) mol/l decreases activity of methemoglobin reductase in human red cells on average by 13.9%. Higher concentrations potentiate this effect. Similar changes in methemoglobin reductase activity were noted after introduction of guanidine-succinic acid into the mixture. This agent in concentration 5.6 x 10(-5) mol/l inhibited activity of the tested enzyme by 34.2% on average. Combined methylguanidine in concentration of 5.4 x 10(-5) mol/l and guanidine-succinic acid in concentration of 2.8 x 10(-5) mol/l inhibited methemoglobin reductase activity by 33.0% on average. It may be suggested, that methylguanidine and guanidine-succinic acid being low molecular uremic toxins may significantly decrease methemoglobin reductase activity in red cells of patients with chronic renal failure.     

Studies on the effect of low-molecule uremic toxins on the activity of glucose-6-phosphate dehydrogenase (E.C.1.1.1.49) and transketolase (E.C.2.2.1.1) in human red blood cells

The effect in vitro was studied of methylguanidine (MG) and guanidinosuccinic acid (GSA) on the activity of glucose-6-phosphate dehydrogenase (G-6-P DH) and transketolase (ETKA) of normal erythrocytes. The results show that MG in concentration (1.8 X 10(-5) mol/l) approximate to or higher then its concentration in plasma of patients with chronic renal failure (ch.r.f.) inhibits the activity of G-6-P DH. The changes of similar nature have been observed in case of GSA. In the same incubation conditions MG or GSA bring about the inhibition of ETKA activity in red blood cells. MG and GSA jointly introduced into incubation mixture in concentrations approximate to those appearing in plasma of patients with ch.r.f. cause the inhibition of G-6-P DH by 35.6% on the average, with no statistically significant differences in ETKA activity.     

Upregulation of angiotensin-converting enzyme by vascular endothelial growth factor

The role of vascular endothelial growth factor (VEGF), a potent endothelium-specific angiogenic factor, in the regulation of angiotensin-converting enzyme (ACE) in cultured human umbilical vein endothelial cells (HUVECs) was studied. VEGF (0.07-1.2 x 10(-6) mmol/l) caused a dose-dependent increase in ACE measured in intact endothelial cells and increased the expression of ACE mRNA. The stimulatory effect of VEGF was inhibited by pretreatment of endothelial cells with the tyrosine kinase inhibitor herbimycin (4.35 x 10(-5) mmol/l). The stimulatory effect of VEGF was potentiated by the selective cGMP phosphodiesterase inhibitor zaprinast (0.1 mmol/l). The nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME; 5.4 mmol/l) suppressed the stimulatory effect of VEGF. The nonselective cyclooxygenase (COX) inhibitor indomethacin (5 microM) and the selective COX-2 inhibitor NS-398 (5 microM) potentiated the stimulatory effect of VEGF, whereas the selective COX-1 inhibitor resveratrol (5 microM) was without effect. ACE induction by VEGF was inhibited by the selective protein kinase C (PKC) inhibitor GF109203X (2.5 x 10(-3) mmol/l) and by downregulating PKC with phorbol 12-myristate 13-acetate. In summary, VEGF induced ACE in cultured HUVECs. Intracellular events such as tyrosine kinase activation, PKC activation, and increase of cGMP were probably involved in ACE induction by VEGF. Nitric oxide may partially contribute to ACE induction by VEGF. The powerful capacity of VEGF to increase ACE in endothelial cells shown here suggests a synergistic relation between VEGF and the renin-angiotensin system in vascular biology and pathophysiology.     

Retinoic acid is a modulator of thyroid hormone activation of Ca2+-ATPase in the human erythrocyte membrane

The thyroid hormones thyroxine (T4) and 3,3',5-L-triiodothyronine (T3) stimulate plasma membrane Ca2+-ATPase (EC 3.6.1.3) activity in human erythrocytes by a mechanism independent of the cell nucleus. The current studies were conducted to determine the effect of retinoic acid on the extranuclear activation by T4 and T3 of Ca2+-ATPase in the human red cell. The retinoid inhibited basal and T4-stimulatable activity of that enzyme in a dose-dependent manner. At the highest tested concentration (10(-6) M), retinoic acid inhibited basal enzyme activity by 25% and T4-stimulated activity by 72%. A concentration as low as 5 x 10(-10) M retinoic acid shifted the dose-response curve of both T4 and T3 so that the concentration of each associated with maximal enzyme stimulation was 10(-9) M instead of 10(-10) M. Retinoic acid displaced [125I]T4 binding to red cell membranes as effectively as unlabeled T4. Retinol failed to influence either basal or T4-stimulated enzyme activity or to displace T4 binding. These results indicate that retinoic acid can partially block the T4 and T3 stimulation of Ca2+-ATPase in human red cell membranes and suggest a physiologic role for the retinoid as a modulator of this peripheral action of thyroid hormone. They suggest that the red cell membrane is an important site of action for this active retinoid.     

Stimulation of pyruvate kinase phosphatase activity by insulin in isolated rat hepatocytes

Addition of insulin (10(-8)M) to hepatocytes, incubated either in the absence or in the presence of a suboptimal concentration of glucagon, caused the reactivation of pyruvate kinase and simultaneously provoked a transient stimulation of pyruvate kinase phosphatase activity (40-70% over control values). The stimulatory effect of insulin on pyruvate kinase phosphatase activity was dose-dependent (ED50 = 1 to 2 X 10(-11)M) and persisted after Sephadex G-25 filtration or ammonium sulfate precipitation of hepatocytes extracts. Our results demonstrate that insulin exerts a short-term regulation on hepatic pyruvate kinase phosphatase activity.     

Factors affecting the activity of pyruvate kinase of Acetobacter xylinum

1. Extracts of Acetobacter xylinum were found to contain the glycolytic enzymes involved in the conversion of triose phosphate into pyruvate. Pyruvate kinase had the lowest relative activity. Phosphofructokinase activity was not detected in the extracts. 2. Only slight differences in the activity of pyruvate kinase were observed between cells grown on glucose and those grown on intermediates of the tricarboxylic acid cycle. 3. Pyruvate kinase, partially purified from ultrasonic extracts by ammonium sulphate fractionation, required Mg(2+) ions for activity. It was not activated by K(+) or NH(4) (+) ions. 4. The plots representing the relationship between initial velocity and phosphoenolpyruvate concentration were sigmoidal, suggesting a co-operative effect for phosphoenolpyruvate. The Hill coefficient (n) for phosphoenolpyruvate was 2. The rate of the reaction changed with increasing ADP concentrations according to normal Michaelis-Menten kinetics. 5. The enzyme was inhibited by ATP (K(i)0.9x10(-3)m). The inhibition was competitive with regard to ADP but not with regard to phosphoenolpyruvate. It was not relieved by excess of Mg(2+) ions. 6. The possible relationship of the properties of pyruvate kinase to regulatory mechanisms for controlling gluconeogenesis and carbohydrate oxidation in A. xylinum is discussed.     

Inhibitory effect of antipsychotic drugs on the Con A- and LPS-induced proliferative activity of mouse splenocytes: a possible mechanism of action.

Antipsychotic drugs are widely used to alleviate a number of psychic disorders and have been found to modulate some immune parameters, but the molecular mechanism of their action on the proliferative activity has been poorly recognized. In the present study, we investigated effects of various antipsychotics on the proliferative activity of lymphocytes stimulated by concanavalin A (Con A) and lipopolysaccharide (LPS). Chlorpromazine (3 x 10(-6)-10(-4) M) showed the most potent effect in inhibiting 3H-thymidine incorporation into C57BL/6 mouse spleen cells stimulated by Con A and LPS. Treatment of the cells with thioridazine (10(-5)-10(-4) M), promazine (10(-5)-10(-4) M), haloperidol (10(-5)-10(-4) M), risperidone (10(-5)-10(-4) M), raclopride (3 x 10(-5) - 10(-4) M), remoxipride (3 x 10(-5)-10(-4) M) and clozapine ( 3 x 10(-5)-10(-4) M), but not with sulpiride (10(-7)-10(-4) M), suppressed proliferative activity of splenocytes after Con A stimulation. On the other hand, LPS-induced proliferation of splenocytes was inhibited by clozapine, promazine, thioridazine and haloperidol, but not by risperidone, remoxipride, sulpiride and raclopride. In the next part of the study, the influence of some kinase modulators on chlorpromazine- and clozapine-evoked inhibition of the proliferative activity of splenocytes was determined. Wortmannin, a selective phosphatidylinositol 3-kinase (PI3-K) inhibitor, blocked chlorpromazine and clozapine inhibitory effect on the mitogen-stimulated splenocyte proliferation. The involvement of PI 3-K /protein kinase B (PKB, Akt) pathway was confirmed by the results of the Western blot study, which showed that both drugs increased the level of active phospho-Ser-473 Akt, without changing the total Akt level, and decreased the level of active, nonphosphorylated glycogen synthase kinase-3 (GSK-3beta). Additionally, we have found that chlorpromazine action was also attenuated by a selective p-38-MAPK inhibitor, while clozapine effect was suppressed by a protein kinase C (PKC) activator. The obtained results indicated that atypical antipsychotic drugs markedly inhibited the proliferative activity of splenocytes only after ConA stimulation. Inhibition of the proliferative capability of splenocytes by chlorpromazine and clozapine resulted mainly from the activation of PI3-K/Akt pathway.     

Control of sulfatase and sulfotransferase activities by medrogestone in the hormone-dependent MCF-7 and T-47D human breast cancer cell lines.

In the present study, we explored the effect of the progestin medrogestone on the sulfatase and sulfotransferase activities in the hormone-dependent MCF-7 and T-47D human breast cancer cell lines. After 24 h incubation at 37 degrees C of physiological concentrations of estrone sulfate ([3H]-E1S: 5x10(-9) mol/l), it was observed that this estrogen was converted in a great proportion to E2 in both cell lines. Medrogestone significantly inhibits this transformation, at all the concentrations tested (5x10(-8) to 5x10(-5) mol/l), in both cell lines. The IC50 values were 1.93 micromol/l and 0.21 micromol/l in MCF-7 and T-47D cells, respectively. In another series of studies, after 24 h incubation at 37 degrees C of physiological concentrations of estrone ([3H]-E1: 5x10(-9) mol/l), the sulfotransferase activity was detectable in both cell lines. Estrogen sulfates (ES) are found exclusively in the culture medium, which suggests that as soon as they are formed they are excreted into the medium. Medrogestone has a biphasic effect on sulfotransferase activity in both cell lines. At low doses: 5x10(-8) and 5x10(-7) mol/l, this compound stimulates the enzyme by +73.5 and 52.7%, respectively, in MCF-7, and by 84.5 and 62.6% in T-47D cells. At high concentrations: 5x10(-6) and 5x10(-5) mol/l, medrogestone has no effect on MCF-7 cells, but inhibits the sulfotransferase activity in T-47D cells by -31.4% at 5x10(-5) mol/l. In conclusion, the inhibitory effect provoked by medrogestone on the enzyme involved in the biosynthesis of E2 (sulfatase pathway) in estrogen-dependent breast cancer, as well as the stimulatory effect on the formation of the inactive ES, support a probable anti-proliferative effect of this progestin in breast tissue. Clinical applications of these findings can open new therapeutic possibilities for this disease.     

Urea inhibition of renal (NA+ + K+)ATPase activity is reversed by cAMP

In the present work we studied the modulation of the effect of urea on the renal (Na+ + K+)ATPase by cAMP. We observed that urea inhibits the (NA+ + K+)ATPase activity in a dose-dependent manner, reaching 60% of inhibition at the concentration of 1M. This effect was completely reversed by dibutyryl-cAMP (dBcAMP) at 5 x 10(-4)M. The effect of dBcAMP was mimicked by 50 units of the catalytic subunit of protein kinase A and completely abolished by 5 x 10(-7)M H89, an inhibitor of protein kinase A. Addition of 1M urea decreases basal phosphorylation of the immunoprecipitated (NA+ + K+)ATPase in 50%, with this effect completely reversed by 5 x 10(-4)M dBcAMP. Furthermore, 5 x 10(-4)M dBcAMP by itself induced (NA+ + K+)ATPase phosphorylation. Taken together these data indicate that cAMP could be, in addition to the organic solutes already known, an important physiological modulator of the deleterious effect of urea on enzyme activity.     

Pancreatic islets contain the M2 isoenzyme of pyruvate kinase its phosphorylation has no effect on enzyme activity

To determine which of the major isoenzymes of pyruvate kinase pancreatic islet pyruvate kinase most resembled, it was compared to pyruvate kinase from other tissues in kinetic and immunologic studies. The pattern of activation by fructose bisphosphate and the patterns of inhibition by alanine and phenylalanine were most similar to those of the M₂ isoenzyme from kidney and were dissimilar to those of the isoenzymes from skeletal muscle (type M₁) and liver (type L). The islet pyruvate kinase was inhibited by anti-M₁ pyruvate kinase serum (which crossreacts with the M₂ isoenzyme), but not by anti-L pyruvate kinase. These results are most consistent with islets possessing predominantly, if not exclusively, the M₂ isoenzyme of pyruvate kinase. We previously showed that rat pancreatic islet cytosol contains protein kinases that can catalyze a calcium-activated phosphorylation of an endogenous peptide that has properties, such as subunit molecular weight and isoelectric pH, that are identical to those of the M₂ and M, isoenzymes of pyruvate kinase, and that islet cytosol can catalyze phosphorylation of muscle pyruvate kinase. In the present study it was shown that incubating islet cytosol with ATP under conditions known to permit phosphorylation and inhibition of liver pyruvate kinase did not affect the islet pyruvate kinase activity. It is concluded that phosphorylation of the islet pyruvate kinase has no immediate effect on enzyme activity.Abbreviations EGTA ethylene glycos his (-aminoethyl ether)-N,N,NN-tetraacetic acid - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid     

Ouabain-insensitive Na(+)-ATPase activity is an effector protein for cAMP regulation in basolateral membranes of the proximal tubule

This study describes the modulation of the ouabain-insensitive Na(+)-ATPase activity from proximal tubule basolateral membranes by cAMP. An increase in dibutyryl-cAMP (d-cAMP) concentration from 10(-8) to 5x10(-5) M stimulates the ouabain-insensitive Na(+)-ATPase activity. The ATPase activity increases from 6.0+/-0.4 to 10.1+/-0.7 nmol Pi mg(-1) min(-1), in the absence and presence of 5x10(-6) M d-cAMP, respectively. Similarly, the addition of cholera toxin (CTX), forskolin (FSK) or guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) also increases the Na(+)-ATPase activity in a dose-dependent manner, with maximal effect at 10(-8) M, 10(-6) M and 10(-7) M, respectively. The effect of 10(-8) M CTX is not additive to the effect of GTPgammaS, and is completely abolished by 200 microM guanosine 5'-O-(2-thiodiphosphate). The stimulatory effects of CTX and FSK on the Na(+)-ATPase activity are accompanied by an increase in cAMP formation by the basolateral membranes of the proximal tubule cells. Furthermore, 10(-8) M protein kinase A peptide inhibitor (PKAi) completely abolishes the stimulatory effect of 5x10(-6) M d-cAMP or 10(-4) M FSK on the Na(+)-ATPase activity. Incubation of the basolateral membranes with [gamma-(32)P]ATP in the presence of d-cAMP or FSK increases the global hydroxylamine-resistant phosphorylation and especially promotes an increase in phosphorylation of protein bands of approximately 100 and 200 kDa. This stimulation is not seen when 10(-8) M PKAi is added simultaneously. Taken together these data suggest that activation of a cAMP/PKA pathway modulates the Na(+)-ATPase activity in isolated basolateral membranes of the proximal tubule.     

An in vitro novel mechanism of regulating the activity of pyruvate kinase M2 by thyroid hormone and fructose 1, 6-bisphosphate

We have recently shown that the cytosolic thyroid hormone binding protein (p58-M2) in human epidermoid carcinoma A431 cells is a monomer of pyruvate kinase, subtype M2 (PKM2). To characterize further the molecular properties of p58-M2, we overexpressed p58-M2 in Escherichia coli and purified it to homogeneity. At 22 degrees C, the monomeric p58-M2, exhibited kinase activity with an apparent Vmax of 22 +/- 9 units/mg. The Km for adenosine diphosphate (ADP) and phosphoenolpyruvate (PEP) are 3.85 +/- 2.4 and 1.55 +/- 0.73 mM, respectively. Upon activation by fructose 1,6-bisphosphate (Fru-1,6-P2), Vmax and Km for ADP and PEP were changed to 490 +/- 27 units/mg and 0.63 +/- 0.09 and 0.13 +/- 0.01 mM, respectively. These results indicated that p58-M2 has intrinsic kinase activity. Analysis of the molecular size indicated that the activation of p58-M2, by Fru-1,6-P2 resulted in the association of the monomeric p58-M2 to the tetrameric PKM2. p58-M2 bound to 3,3',5-triiodo-L-thyronine (T3) (Ka = 1.7 x 10(7) M-1) and exhibited analogue specificity, whereas PKM2 did not bind thyroid hormone. The order of binding affinity was L-T3 greater than L-thyroxine greater than 3,3',5-triiodothyropropionic acid greater than 3'-isopropyl-3,5-triiodo-L-thyronine greater than 3'5',3-triiodo-L-thyronine. Binding of T3 and its analogues resulted in the inhibition of the kinase activity of p58-M2. The order of kinase inhibitory activity and preventing its association to tetrameric PKM2 was parallel to that of binding activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of transketolase from human erythrocytes

Transketolase was isolated from human red blood cells with over 6,200 fold purification by a new method. The stepwise procedure for the isolation of the enzyme from erythrocyte hemolysate included the use of ethanol/chloroform precipitation, chromatography on hydroxyapatite and finally, affinity adsorption on carboxymethyl-cellulose. The molecular weight of erythrocyte transketolase, as determined by polyacrylamide gel electrophoresis, appeared to be about 140,000. The pH optimum for activity was between 7.6 and 7.8 and the optimum temperature for activity was 50 degrees C. The Km values for xylulose-5-phosphate, ribose-5-phosphate and fructose-6-phosphate were 2.0 x 10(-4) M, 3.2 x 10(-4) M and 2.0 x 10(-3) M, respectively.     

Norelgestromin as selective estrogen enzyme modulator in human breast cancer cell lines. Effect on sulfatase activity in comparison to medroxyprogesterone acetate

Human breast cancer tissue contains enzymes (estrone sulfatase, 17beta-hydroxysteroid dehydrogenase, aromatase) involved in the last steps of estradiol (E(2)) formation. In this tissue, E(2) can be synthesized by two main pathways: (1) sulfatase-transforms estrogen sulfates into bioactive E(2), and the (2) aromatase-converts androgens into estrogens. Quantitative assessment of E(2) formation in human breast tumors indicates that metabolism of estrone sulfate (E(1)S) via the sulfatase pathway produces 100-500 times more E(2) than androgen aromatization.In the present study, we demonstrated in T-47D and MCF-7 human breast cancer cells that norelgestromin (NGMN) (a metabolite of norgestimate) is a potent inhibitory agent of the estrone sulfatase activity. After 24h incubation of physiological concentrations of E(1)S (5 x 10(-9)mol/l) the inhibitory effect of NGMN at concentrations of 5 x 10(-9), 5 x 10(-7) and 5 x 10(-5)mol/l was 43+/-7, 74+/-4 and 97+/-2%, respectively, in T-47D cells; 25+/-4, 57+/-5 and 96+/-2% respectively, in MCF-7 cells. Comparative studies using medroxyprogesterone acetate (MPA) showed that this progestin also has an inhibitory effect on sulfatase activity, but significantly less intense than that of NGMN. The inhibition for MPA at concentrations of 5 x 10(-9), 5 x 10(-7) and 5 x 10(-5)mol/l was 31+/-5, 47+/-3 and 61+/-3%, respectively, for T-47D cells; 6+/-3, 20+/-3 and 63+/-4%, respectively, for MCF-7 cells.In conclusion, the present data show that NGMN is a very potent inhibitory agent for sulfatase activity in the hormone-dependent breast cancer cells, resulting in decreased tissue concentration of E(2). The clinical significance of this finding remains to be elucidated.     

Brain pyridoxal kinase: photoaffinity labeling of the substrate-binding site

4-Benzoylbenzoic acid inhibits pyridoxal kinase activity competitively with respect to pyridoxal. The Ki was determined to be 5 x 10(-5) M. Binding studies showed that 4-benzoylbenzoic acid bound to pyridoxal kinase at a 1:1 molar ratio and with a dissociation constant (Kd) of 5.9 x 10(-5) M. Photoirradiation of pyridoxal kinase in the presence of a 10-fold excess of 4-benzoylbenzoic acid at pH 6.5 resulted in an irreversible loss of enzymatic activity; this photoinactivation was prevented by the presence of pyridoxal. Amino acid analysis revealed that 1 tyrosine residue/subunit was modified during photoinactivation. The presence of a tyrosine residue at the active site of pyridoxal kinase was confirmed by reaction with tetranitromethane. In the presence of 1 x 10(-4) M tetranitromethane, a complete loss of the kinase activity was observed after incubation at 25 degrees C for 8 min, with modification of a total of 3 tyrosine residues. The second-order rate constant (K2) of the reaction between the tyrosine residues and tetranitromethane was determined to be 53.3 s-1 M-1.     

Inhibition of human gastric H(+)-K(+)-ATPase alpha-subunit gene expression by Helicobacter pylori

Clinical studies and in vitro data from isolated parietal cells suggest that acute Helicobacter pylori infection inhibits acid secretion. Gastric acidification is mediated by H(+)-K(+)-ATPase, an integral protein of parietal cell apical membranes. To test the hypothesis that H. pylori downregulates H(+)-K(+)-ATPase alpha-subunit (HKalpha) gene expression and to identify potential intracellular signaling pathways mediating such regulation, we transfected human gastric adenocarcinoma (AGS) cells with human and rat HKalpha 5'-flanking DNA fused to a luciferase reporter plasmid. Histamine caused dose-dependent, cimetidine-sensitive (10(-4) M) increases in cAMP, free intracellular Ca(2+), and HKalpha promoter activation in AGS cells. H. pylori infection of transfected AGS cells dose dependently inhibited basal and histamine-stimulated HKalpha promoter activity by 80% and 66%, respectively. H. pylori dose dependently inhibited phorbol myristate acetate-induced (10(-7) M) and staurosporine- (10(-7) M) and calphostin C-sensitive (5 x 10(-8) M) activation of HKalpha promoter. Also, H. pylori inhibited epidermal growth factor (EGF) (10(-8) M), genistein-sensitive (5 x 10(-5) M) activation of HKalpha promoter, reducing activity to 60% of basal level. These data suggest that H. pylori inhibits HKalpha gene expression via intracellular pathways involving protein kinases A and C and protein tyrosine kinase, AGS cells have functional histamine H(2) and EGF receptors, and transiently transfected AGS cells are a useful model for studying regulation of HKalpha gene expression.     

Hypocrellin and hypericin-induced phototoxicity of HL-60 cells: apoptosis or necrosis?

Hypericin and hypocrellin are potential antiviral and antineoplastic agents with multiple modes of light-induced biological activity connected with a production of singlet oxygen and/or excited-state proton transfer and consequent pH drop formation in the drugs environment. In present work light-induced cytotoxicity of hypericin and hypocrellin and mechansim of cell death (apoptosis or necrosis) on human leukemic cell line HL-60 was studied. As a mean for apoptosis detection we used poly (ADP-ribose) polymerase (PARP) as a sensitive marker of early stages of apoptosis. Our results show that exposition of HL-60 cells to hypericin (1 x 10(-5) mol x l(-1)) for 4 hours has no effect on PARP cleavage. However, after 24 and 48 hours of illumination there is evident that hypericin in this concentration cleaved PARP (116 kDa) into two fragments (85 and 25 kDa). Contrary to hypericin, hypocrellin in concentration 1 x 10(-5) mol x l(-1) after 4 hours of illumination cleaved PARP into two fragments typical for apoptosis. In lower concentration (1 x 10(-6) mol x l(-1)) hypocrellin possess also significant cytotoxic activity. Because we detected no fragmentation of PARP in all observed time periods we suggest that cytotoxic effect of hypocrellin in this concentration is due to induction of necrosis. Our results support the hypotesis that the hypericin and hypocrellin has similar mechanism of action and illumination increases cytotoxic effect of both agents.     

Effect of Medrogestone on 17beta-hydroxysteroid dehydrogenase activity in the hormone-dependent MCF-7 and T-47D human breast cancer cell lines

Estradiol (E2) is one of the most important hormones supporting the growth and evolution of breast cancer. Consequently, to block this hormone before it enters the cancer cell, or in the cell itself, has been one of the main targets in recent years. In the present study we explored the effect of Medrogestone (Prothil) on 17beta-hydroxysteroid dehydrogenase (17beta-HSD) activities of the hormone-dependent MCF-7 and T-47D human breast cancer cell lines. Using physiological doses of estrone ([3H]-E1: 5 x 10(-9) mol/l) this estrogen is converted in a great proportion to E2 in both cell lines. After 24 h of the cell culture, Medrogestone significantly inhibits this transformation in a dose-dependent manner by 39% and 80% at 5 x 10(-8) M and 5 x 10(-5) M, respectively in T-47D cells; the effect is less intense in MCF-7 cells: 25% and 55% respectively. The IC50 values are 0.45 micromol/l in T-47D and 17.36 micromol/l in MCF-7 cells. It is concluded that the inhibition provoked by Medrogestone on the reductive 17beta-HSD activity involved in the local biosynthesis of the biologically active estrogen estradiol, may constitute a new therapeutic approach for the treatment of breast cancer.     

Modulation of the (Na(+)+K+)ATPase activity by Angiotensin-(1-7) in MDCK cells

In the present paper the effect of Ang-(1-7) on the distal tubule (Na(+)+K+)ATPase activity was evaluated by using MDCK cells as a model. Confluent cell monolayers were incubated with increasing concentrations of Ang-(1-7) for 30 min. Thereafter, the (Na(+)+K+)ATPase activity was evaluated and a dose-dependent (from 10(-12) to 10(-7) M) inhibition was observed. The maximal inhibitory effect (54%) was reached at the concentration of 10(-8) M. The inhibitory effect of Ang-(1-7) was not affected by the AT2 receptor selective antagonist PD123319 (from 10(-10) to 10(-7) M) but was blocked in a dose-dependent manner by the AT1 receptor selective antagonists losartan (10(-10) M), candesartan (10(-17) M), irbesartan (2 x 10(-12) M) and telmisartan (2 x 10(-16) M). The signaling pathway triggered by stimulation of the AT(1) receptor was also investigated. The PI-phospholipase C (PI-PLC) inhibitor U73122 (5 x 10(-8) M) blocked the inhibitory effect elicited by Ang-(1-7). Involvement of the protein kinase C (PKC) was evidenced by the sensitivity of the inhibitory effect of Ang-(1-7) to calphostin C (6.32 x 10(-7) M) and the lack of additive effects when the cells were co-incubated with Ang-(1-7) and 3.2 x 10(-8) M PMA. Altogether, these results demonstrate that Ang-(1-7) inhibits the (Na(+)+K+)ATPase activity of the prototypic distal tubule cell MDCK through the AT1 receptor-mediated stimulation of PI-PLC/PKC signaling pathway.     

Inactivation of phosphofructokinase by glucagon in rat hepatocytes.

Kinetic evidence of a time- and dose-dependent inactivation of phosphofructokinase by glucagon in isolated rat hepatocytes is reported. This inactivation, which persists after gel filtration of a cell-free extract on Sephadex G-25 and after 400-fold purification of the enzyme on agarose-ATP, is observed when the enzyme activity is measured at subsaturating concentrations of fructose 6-phosphate, while there is no change in Vmax. Phosphofructokinase inactivation by glucagon parallels the known inactivation of pyruvate kinase L and activation of glycogen phosphorylase alpha. Exogenous cyclic AMP mimics the effect of this hormone. Half-maximal effect for both phosphofructokinase and pyruvate kinase L is caused by a similar dose of glucagon (1 x 10(-10) M). The inactivation of phosphofructokinase by nonsaturating concentration of glucagon is reversed spontaneously within 40 min of incubation and this reversion is accelerated by insulin.