Structural specificity of the NaK-ATPase inhibition by sanguinarine, an isoquinoline benzophenanthridien alkaloid.
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Alkaloids↗
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Binding of sanguinarine to deoxyribonucleic acids of differing base composition.
The binding of the alkaloid sanguinarine to natural DNAs of differing GC content has been studied by spectrophotometry and viscometry techniques. Binding parameters determined from spectrophotometric measurements by Scatchard analysis, according to an excluded-site model, indicate a very high specificity of sanguinarine binding to GC rich DNA. In the strong binding region, the increase of contour length of DNA depends strongly on its base composition, being larger with GC rich DNA than with AT rich DNA. It is concluded that the alkaloid binds preferentially to the GC pairs in DNA template.
Alkaloids↗
Effects of the antitumor drugs 3-nitrobenzothiazolo[3,2-alpha]quinolinium and fagaronine on nucleic acid and protein synthesis.
3-Nitrobenzothiazolo[3,2-alpha]quinolinium perchlorate (NBQ) has been shown to be active against in vivo experimental tumors of P388 and Ehrlich ascites cells. Furthermore, it has been established that NBQ binds to DNA by intercalation. In this work we describe its effects on DNA, RNA and protein syntheses both in KB cells and in cell-free synthesizing systems. Fagaronine, an alkaloid structurally related to NBQ, was studied also in an attempt to establish the basis for future studies on structure-activity relationships. Both NBQ and fagaronine inhibited DNA, RNA and protein syntheses in KB cells, with essentially equal effectiveness. Exposure of KB cells to NBQ for 2 hr caused irreversible inhibition of DNA, RNA and protein syntheses. Studies in cell-free systems showed that NBQ strongly inhibited Escherichia coli DNA polymerase I, whereas RNA polymerase activities were moderately affected. Furthermore, both drugs inhibited protein synthesis in cell-free systems derived from rabbit reticulocytes and Saccharomyces cerevisiae. Our results indicate that NBQ and fagaronine exert their cytotoxic activity by at least two independent mechanisms: inhibition of DNA activity by binding to this molecule, and inhibition of protein synthesis probably by interacting with the ribosomal system.
Alkaloids↗
The antileukemic alkaloid fagaronine is an inhibitor of DNA topoisomerases I and II.
The antileukemic alkaloid, fagaronine, is a potent differentiation inducer of various hematopoietic cell lines. We show here that fagaronine is a DNA base-pair intercalator with a K(app) of 2.1 x 10(5) M-1 for calf thymus DNA. Fagaronine inhibits the catalytic activity of purified calf thymus topoisomerase I as shown by relaxation of supercoiled plasmid DNA followed by electrophoresis in neutral as well as in chloroquine-containing gels. The catalytic activity of topoisomerase I is inhibited at concentrations above 30 microM. Fagaronine also inhibits the catalytic activity of purified calf thymus topoisomerase II at concentrations above 25 microM as shown by decatenation of kinetoplast DNA. Fagaronine stabilizes the covalent DNA-enzyme reaction intermediate (the cleavable complex) between topoisomerase I and linear pBR322 DNA at concentrations up to 1 microM. Further increase of the fagaronine concentration leads to a progressive decrease in the cleavable complex formation, which is totally inhibited at 100 microM. In contrast, up to 1 microM fagaronine has no effect on cleavable complex formation between purified calf thymus topoisomerase II and linear pBR322 DNA, whereas cleavable complex formation is inhibited at higher concentrations. Exposure to fagaronine results in an increase in DNA-protein complex formation in intact P388 murine leukemia cells. P388CPT5 cells, which have an altered topoisomerase I activity, are 4-fold resistant to the growth inhibitory effects of fagaronine compared to the parental cell line. Similarly, DC-3F/9-OH-E Chinese hamster fibrosarcoma cells, which have an altered topoisomerase II activity, are about 5-fold resistant to the growth inhibitory effects of fagaronine. We conclude that fagaronine is an inhibitor of both DNA topoisomerase I and II and propose that this might play a role in the cytotoxic activity.
Alkaloids↗
Association between leukotriene B4-induced phospholipase D activation and degranulation of human neutrophils.
We have explored the role of phospholipase D (PLD) activation in leukotriene B4 (LTB4)-induced Ca2+ mobilization and degranulation of human neutrophils. Stimulation of [3H]alkyl-acyl-phosphatidylcholine-labeled neutrophils with LTB4 resulted in a rapid accumulation of [3H]alkyl-phosphatidic acid (PA) as well as a somewhat slower accumulation of [3H]alkyl-diglyceride (DG). In the presence of ethanol, PLD catalyzed a transphosphatidylation reaction in which LTB4 increased [3H]alkyl-phosphatidylethanol formation and simultaneously decreased LTB4-induced PA and DG accumulation. This pattern of lipid metabolism is consistent with the conclusion that LTB4 stimulates PLD activity in human neutrophils. Additional studies in which the extracellular and intracellular concentrations of Ca2+ were varied indicated that maximal LTB4-induced PLD activation was dependent upon Ca2+ and potentiated by inhibitors of protein kinase C. The time-course and concentration-response curves for LTB4-induced PLD activation were different from those for LTB4-induced Ca2+ mobilization, as measured by fura-2 fluorescence. On the other hand, the concentration-response curve for LTB4-induced PLD activation was similar to that for LTB4-induced degranulation. Preincubation of the cells with ethanol inhibited LTB4-induced PA and DG accumulation, as well as degranulation, suggesting that one or both of these metabolites were important for this response. In contrast, ethanol had no effect on LTB4-induced Ca2+ mobilization. Propranolol, an inhibitor of phosphatidate phosphohydrolase, abolished DG accumulation in response to LTB4 but had no effect on degranulation, suggesting that PA is more important than DG as a mediator of degranulation. Taken collectively, these data indicate that LTB4-induced activation of PLD in human neutrophils is mediated by a Ca(2+)-dependent mechanism, but not by protein kinase C. In addition, PLD activation in these cells may induce degranulation, but not Ca2+ mobilization.
Alkaloids↗
Interaction of sanguinarine iminium and alkanolamine form with calf thymus DNA.
The interaction of sanguinarine iminium form (structure I) and sanguinarine alkanolamine form (structure II) with calf thymus DNA has been studied in buffer of pH 5.2 and pH 10.5, respectively, where the physicochemical properties of DNA remain unchanged. The binding of sanguinarine iminium form to DNA is characterized by hypochromism and bathochromism in the absorption band, quenching of fluorescence intensity, increase in fluorescence polarization anisotropy, increase in positive and negative ellipticity of DNA, sign and magnitude of the thermodynamic parameters and increase in contour length of sonicated rodlike duplex DNA indicating that it binds to DNA by a mechanism of intercalation. In contrast, sanguinarine alkanolamine form does not show (i) any significant change in fluorescence polarization anisotropy, (ii) alteration of B form structure of DNA and (iii) increase in contour length of DNA indicating that it does not bind to DNA. But at a very high concentration of DNA, the alkanolamine form is influenced to form an iminium-DNA complex.
Alkaloids↗
Dependence of endothelin-1 secretion on Ca2+.
The role of Ca2+ and protein kinase C (PKC) activity in the release of immunoreactive endothelin-1 (ET-1) from cultured porcine aortic endothelial cells of first or second passage has been studied. ET-1 accumulation within cells and secretion into cell-conditioned medium over 3 and/or 5 hr was measured. Confluent cells incubated in medium containing 1.8 mM Ca2+ (control condition) accumulated and released ET-1 in a time-dependent way. Reducing intracellular free Ca2+ concentration ([Ca2+]i) by adding the Ca2+ entry blockers NiCl2 (0.5 mM) and amiloride (1 mM) or the Ca2+ chelator EGTA (5 mM) to the incubation medium reduced ET-1 secretion to between 50 and 30% of controls, respectively (P < 0.01). To determine the effect of high [Ca2+]i on ET-1 release, cells were incubated with thapsigargin (10-1000 nM) or Ca2+ ionophore A23187 (1 microM) which raised [Ca2+]i progressively from 190 nM (control) to > 1 microM. Both agents reduced ET-1 secretion in a concentration-dependent manner to between 50 and 20% of controls (P < 0.01). Intracellular levels of ET-1 were also reduced at both low and high [Ca2+]i (P < 0.01). In the presence of the PKC inhibitors chelerythrine (50 microM) and H-7 (60 microM), basal ET-1 secretion was reduced to below 20% of controls (P < 0.01). The PKC activator phorbol 12-myristate 13-acetate (0.4 microM) stimulated ET-1 release 1.4-fold (P < 0.01) and its effect was abolished by EGTA (5 mM). Increased [Ca2+]i stimulated the production and release of cyclic guanosine-3',5'-monophosphate, but basal ET-1 secretion rates correlated poorly with nucleotide levels. These data indicate that: (i) at resting [Ca2+]i concentrations, ET-1 release is close to maximal and is reduced at lower and higher concentrations, resulting in a bell-shaped relationship between [Ca2+]i and ET-1 release; and (ii) basal ET-1 release is largely determined by Ca(2+)-dependent PKC activity.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
Paradoxical stimulatory effect of the kinase inhibitor chelerythrine on the phosphorylation of a approximately 20 K M(r) protein present in the mitochondrial fraction of rat retina.
In order to characterize the phosphorylation of a approximately 20k M(r) protein present in the mitochondrial fraction of the rat retina, chelerythrine chloride, a well-known protein kinase C inhibitor, was tested for activity. Instead of the expected inhibition of the kinase reaction by chelerythrine the phosphorylation of the approximately 20k M(r) protein was stimulated by a factor of 3 at 150 microM. This unique stimulatory action of chelerythrine could be eliminated by the addition of 10 mM dithiothreitol. A suggested mechanism of action for dithiothreitol in the elimination of the increased phosphorylation of the approximately 20k M(r) protein by chelerythrine is the addition of the thiol group of dithiothreitol to the iminium bond of chelerythrine. Taurine, a known inhibitor of the phosphorylation of retinal proteins was also tested in combination with chelerythrine for its effects on the phosphorylation of the approximately 20k M(r) protein. A non-competitive relationship was observed when chelerytrhine was used as the variable activator and taurine as the fixed inhibitor (30 mM). The stimulatory effect of chelerythrine on the phosphorylation of proteins was not limited to retinal tissue but was also observed in the P2 fraction of brain cortex. Chelerythrine demonstrated only inhibitory effects on the phosphorylation of proteins in a heart mitochondrial fraction.
Alkaloids↗
Neuroprotective effects of PKC inhibition against chemical hypoxia.
The effect of potassium cyanide-induced chemical hypoxia on protein kinase C (PKC) translocation and cell injury was studied in differentiated PC12 cells. The cellular distribution of PKC in control cells and cells exposed to 100 microM and 1 mM KCN for 30 min. was visualized by use of an anti-PKC antibody and confocal laser scanning microscope. In control differentiated PC12 cells, PKC was localized perinuclearly, while following 12-phorbol 13-myristate acetate (PMA) or KCN it was translocated to the plasma and organelle membranes. Western blot analysis was used to quantify the translocation. Chemical hypoxia increased the membrane-bound PKC to 210% of control levels, while chelerythrine, a PKC inhibitor, and block of calcium influx into the cells (with calcium channel blocker and calcium-free medium) prevented this effect. Cyanide-induced PKC translocation persisted for at least 120 min. Cell injury was monitored by measuring lactate dehydrogenase (LDH) efflux from the cells 24 hr after addition of cyanide. PKC activation plays a role in hypoxic damage, since PKC down-regulation (by overnight exposure to PMA) or inhibition (with chelerythrine or staurosporine) conferred protection against KCN-induced cytotoxicity. Ca2+ channel blocker nifedipine also protected against chemical hypoxia. None of the pretreatments rendered complete protection against cyanide-induced hypoxia, indicating that PKC-independent mechanism(s) are also activated during chemical hypoxia and contribute to cell injury.
Alkaloids↗
Protein kinase C and mouse sciatic nerve regeneration.
We have studied the role of protein kinase C (PKC) in peripheral nerve regeneration by using the cultured adult mouse sciatic nerve, which displays regrowth of sensory axons under serum-free conditions. By the use of immunohistochemistry we show that one of the isoforms of PKC, PKC beta, is present in the nerve cell bodies of normal nerves and is upregulated after injury. In spite of this, the specific PKC inhibitor chelerythrine at 5 microM, a concentration well above its IC50 value for PKC, failed to reduce the outgrowth distance of new axons. This was not due to impermeability of the drug, since the same concentration caused a clear reduction of the injury-induced proliferation of Schwann cells in the crush region. Likewise, HA-1004, an inhibitor of cyclic nucleotide-dependent protein kinases, also lacked effect on outgrowth when used on its own, even at very high concentrations (100 microM). In contrast, outgrowth was significantly reduced when 5 microM chelerythrine and 5 microM HA-1004 were used in combination. In conclusion, the present results suggest that PKC-activity is important but not indispensable for the regeneration process. Successful completion of the latter could be achieved by several, perhaps redundant, phosphorylation systems.
Alkaloids↗
2,3-Butanedione monoxime modifies the glycine-gated chloride current of acutely isolated murine hypothalamic neurons.
In this study, we explored the effect of the chemical phosphatase 2,3-butanedione monoxime (BDM) on glycine current (IGly) of murine ventromedial hypothalamic neurons. Co-application of 0.01 to 67 mM BDM increased IGly decay rate with little change of the peak amplitude. This effect was both rapid in onset and offset and required the presence of the agonist. Pretreatment with BDM alone did not alter-IGly decay. In addition, dialysis of neurons with 500 microM ATP-gamma-S did not alter the acute effect of BDM. Thus, this effect may result from open channel block rather than BDM-induced dephosphorylation of the receptor/channel protein. In contrast to the acute effect described above, relatively prolonged (i.e., greater than 80 s) pretreatment with BDM reduced peak IGly. The phorbol ester (PDBu), a protein kinase C (PKC) activator, mimicked this effect of BDM. Furthermore, chelerythrine, a specific PKC inhibitor, prevented this effect of BDM on peak IGly. Thus, activation of PKC may mediate this attenuating effect of BDM on IGly. For a sub-population of these pretreated neurons, there was a subsequent potentiation of IGly which followed the initial suppressant effect. This potentiation may be due to a phosphatase effect of BDM, since it was observed more frequently when neurons were also pretreated with the protein kinase inhibitors H7 or chelerythrine. These findings suggest that BDM alters protein kinase activity and acts as a phosphatase to regulate the activity of the glycine receptor/channel complex.
Alkaloids↗
Interaction of the antileukemic alkaloid, 2-hydroxy-3,8,9-trimethoxy-5-methylbenzo[c]phenanthridine (fagaronine), with nucleic acids.
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Alkaloids↗
Chronic inhibition of intracellular Ca2+ release or protein kinase C activation significantly reduces the development of morphine dependence.
We have previously shown that chronic antagonism of metabotropic glutamate receptors in the brain attenuates naloxone-precipitated withdrawal symptoms in rats treated chronically with subcutaneous (s.c.) morphine. Several subtypes of metabotropic glutamate receptors are directly linked, through a guanine nucleotide regulatory protein, to the phosphatidylinositol (p.i.) second messenger system. In the present investigation, we assessed the effect of inhibiting the products of p.i. hydrolysis on the development of opioid dependence. Thus, concurrently with subcutaneous morphine, we infused intracerebroventricularly (i.c.v.) in rats, various doses of chelerythrine, which selectively inhibits the activation of protein kinase C, and thapsigargin, which inhibits the release of intracellular Ca2+ when given chronically. Both chelerythrine and thapsigargin reduced the severity of naloxone-precipitated abstinence symptoms when infused i.c.v. at a dose of 10 nmol/day. A single injection of either chelerythrine or thapsigargin immediately prior to the precipitation of withdrawal failed to decrease the severity of abstinence symptoms. Our results suggest that by chronically inhibiting activity of the phosphatidylinositol system, the development of morphine dependence can be attenuated.
Alkaloids↗
Sanguinarine: a monofunctional intercalating alkaloid.
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Alkaloids↗
Protein kinase C and phospholipase D activation in rat parotid glands.
We have previously demonstrated that muscarinic and alpha-adrenergic receptors regulated a phospholipase D (PLD) activity in parotid glands. Since phorbol 12-myristate, 13-acetate (PMA) induced production of phosphatidylethanol (PEt), a stable metabolite widely accepted as marker of PLD activation, we have investigated the role of protein kinase C (PKC) in PLD stimulation in parotid acini. We tested PKC inhibitors on PEt formation elicited by PMA, by muscarinic and adrenergic agents. Staurosporine and chelerythrine, which act on the catalytic domain of PKC, did not allow the attribution of a role for PKC in PLD activation. Indeed, staurosporine did not affect PMA-mediated PLD activity and chelerythrine showed an important non-specific effect, independent of PKC inhibition. On the other hand, calphostin C, which acts on the regulatory domain of PKC, affected PMA- and receptor-mediated PLD stimulation. We attributed this effect to PKC inhibition and we suggested PKC involvement in PLD regulation in parotid gland. Since only PKC inhibitor acting on the regulatory part of the enzyme affected PLD activity, we also suggested that PKC could be involved in PLD activation through a pathway independent of the phosphorylation mechanism.
Alkaloids↗
Role of protein kinase C in mediating effects of hydrogen peroxide in guinea-pig ventricular myocytes.
The present study examined the effects of hydrogen peroxide (H2O2) on intracellular calcium transients and unloaded cell shortening in the presence of the protein kinase C (PKC) inhibitors 1-(5-isoquinolinesulfonyl-2-methylpiperazine (H7) or chelerythrine chloride (CHC) or the PKC activator phorbol 12-myristate 13-acetate (PMA). Calcium transient amplitudes and cell shortening were measured simultaneously in single, enzymatically dissociated ventricular myocytes loaded with fura2-AM. Exposure of myocytes to H2O2, 25 microM or 75 microM, for 15 min caused a time- and concentration-dependent increase in calcium transient amplitude, cell shortening and the diastolic 340/380 fluorescence ratio. Significant increases in calcium transient amplitude were observed from 7 to 15 min of superfusion with 25 microM H2O2 and the transient amplitude remained elevated throughout the 10 min washout period. In the presence of 75 microM H2O2, transient amplitude was elevated following 2 min and remained elevated for the remainder of the experiment. Significant increases in cell shortening were also observed from 7 to 15 min in the presence of either 25 or 75 microM H2O2. This effect was reversed upon washout of the lower concentration of H2O2 but persisted during the initial 5 min of washout at the higher concentration. The diastolic 340/380 fluorescence ratio was unaltered in the presence of 25 microM of H2O2, however this parameter was significantly increased from 7 to 15 min following exposure to 75 microM H2O2 and remained elevated throughout the washout period. The H2O2-induced increases in calcium transient amplitude and cell shortening were significantly attenuated in myocytes which were pretreated with either H7 or CHC.(ABSTRACT TRUNCATED AT 250 WORDS)
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
Epidemic dropsy following transcutaneous absorption of Argemone mexicana oil.
Four cases manifesting features characteristic of epidemic dropsy following body massage with contaminated mustard oil are reported. A transcutaneous route of absorption for the toxin (sanguinarine) resulting in epidemic dropsy has not been documented previously in man. Oil used for body massage was found to be adulterated with Argemone mexicana oil, while hydrogenated vegetable fat used for cooking did not reveal any contamination. Diagnosis of the disease was confirmed by establishing the presence of sanguinarine in the urine and serum of all four cases.
Adult↗
Antiplatelet effects of protopine isolated from Corydalis tubers.
Protopine inhibited the aggregation and ATP release of rabbit platelets induced by ADP, arachidonic acid, PAF, collagen and ionophore A23187. Although the platelet aggregation caused by thrombin was not inhibited by protopine (100 micrograms/ml), the release reaction was partially suppressed. In rabbit platelet-rich plasma, protopine also inhibited the platelet aggregation caused by ADP, arachidonic acid, PAF and collagen. The thromboxane B2 formation of washed platelets caused by arachidonic acid, collagen, ionophore A23187 and thrombin was suppressed by protopine. Protopine inhibited the intracellular calcium increase caused by arachidonic acid in quin-2/AM loaded rabbit platelets. In the presence of indomethacin, the intracellular calcium increase caused by collagen and PAF was completely suppressed by protopine, and the intracellular calcium increase caused by thrombin was partially inhibited. The phosphoinositides breakdown caused by collagen and PAF was inhibited by protopine, but that by thrombin was not affected significantly. Protopine did not cause the elevation of cyclic AMP level of platelets. It is concluded that the antiplatelet effects of protopine is due to inhibition on thromboxane formation and phosphoinositides breakdown and then lead to the decrease of intracellular calcium concentration.
Adenosine Triphosphate↗