Biomedical subjects

B H Schmidt

Publications and source records attributed to B H Schmidt.

32 records · Page 2Linked to original sources

PubMed1992

Radiolabelled calmodulin ligands: their low affinity and high lipophilicity may lead to artefacts in binding studies.

In order to characterize the interaction site of a series of putative calmodulin antagonists of the diphenylalkylamine type with calmodulin (CaM), a representative member of this chemical class was radiolabelled. The binding of the selected compound, [3H]-VUF 4576, to calmodulin was studied according to a recently described technique using CaM agarose. However, some peculiar results were obtained: the tight binding of [3H]-VUF 4576 increased in presence of cold VUF 4576, resulting in a high non-specific binding. The unexpected results could readily be explained by a high binding capacity of the labelled compound and the cold ligands to the walls of the test tubes used. Such results were also found when [3H]-chlorpromazine ([3H]-CPZ) was applied. In literature comparable findings have been published. To explain such results the influence of positive cooperativity or irreversible binding has been suggested. We suppose that not only in our study, but also in other published investigations, binding to glass of the radioligand and/or the cold compounds may have had a strong influence. We suggest, therefore, that care should be taken in interpreting non-classical displacement data obtained with ligands which combine a rather low affinity and a high degree of lipophilicity, not only for binding to calmodulin, but for other systems as well.

Benzhydryl Compounds↗

PubMed1991

Polymyxin B is a selective and potent antagonist of calmodulin.

Polymyxin B, a cyclic peptide antibiotic, is considered to be a rather selective antagonist of protein kinase C. This drug is therefore widely used to evaluate the involvement of protein kinase C in cellular processes. In the present study, we investigated the effects of polymyxin B on the activity of calmodulin-dependent cyclic 3':5'-nucleotide phosphodiesterase in vitro. The drug potently inhibited this enzyme (IC50 80 nM in the presence of 500 microM Ca2+), while about 200-fold higher concentrations were required to inhibit protein kinase C to the same extent. Phosphodiesterase inhibition was competitive with respect to Ca2+ and calmodulin. Evidence for the formation of a complex between polymyxin B and calmodulin was obtained by polyacrylamide gel electrophoresis under non-denaturing conditions, and by affinity chromatography of calmodulin on polymyxin B-agarose. We therefore suggest that, at least in vitro, polymyxin B is a potent and selective inhibitor of calmodulin.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1991

Cholinergic inositol phosphate formation in striatal neurons is mediated by distinct mechanisms.

In murine striatal neurons devoid of functional synapses (6 days in vitro) the cholinergic agonists carbachol and arecoline evoked dose-dependent inositol phosphate (InsP) responses with mean log EC50s of -4.1 +/- 0.5 and -4.48 +/- 0.1, respectively. Carbachol (1 mM) and arecoline (1 mM) responses were insensitive to tetrodotoxin, a voltage-sensitive Na+ channel blocker, and were blocked by pirenzepine with relatively low affinity (logIC50 = -5.9 +/- 0.3 for the carbachol response and logIC50 = -5.8 +/- 0.3 for the arecoline response). After synaptogenesis (13 days in vitro) the maximal carbachol effect doubled whereas the arecoline response remained unchanged. This additional effect was sensitive to tetrodotoxin and the voltage-dependent Ca2+ channel blocker, omega-conotoxin. The tetrodotoxin-sensitive carbachol response was blocked by lower concentrations of pirenzepine than the tetrodotoxin-insensitive carbachol response. More than 75% of the InsP response evoked by low concentrations of muscarine (1 and 10 microM) was sensitive to tetrodotoxin whereas only 38% of the InsP response stimulated by 1 mM of muscarine could be blocked by tetrodotoxin. These results suggest that there are at least two different mechanisms (depending on the stage of development), activated most probably by two different muscarinic receptors responsible for the carbachol-induced InsP formation in striatal neurons.

Animals↗

PubMed1991

Effects of tiflucarbine as a dual protein kinase C/calmodulin antagonist on proliferation of human keratinocytes and release of reactive oxygen species from human leukocytes.

Various studies have suggested that calmodulin (CaM) is involved in the pathophysiology of psoriasis. Protein kinase C (PKC) is also accepted as playing a regulatory role in cell proliferation as well as in inflammatory processes. Therefore, we investigated the effects of the known CaM antagonist tiflucarbine (BAY/TVX P 4495) on two cellular systems related to the major clinical symptoms of psoriasis: proliferation of cultured human keratinocytes (HaCa T cell line) and release of reactive oxygen species (ROS) from human polymorphonuclear leukocytes (PMNL). Tiflucarbine inhibited both cellular responses in a dose dependent manner. Furthermore, tiflucarbine directly affected PKC, and may thus be considered to be a dual PKC/CaM antagonist with putative antipsoriatic activity. The effects of tiflucarbine on the different parameters were compared with those of the structurally unrelated dual PKC/CaM inhibitor W-7 and those of the potent PKC inhibitor staurosporine. The potencies of all three compounds were found to be in the same range as their PKC-inhibiting potency. Our data indicate that PKC, rather than CaM, may play a regulatory role in the release of ROS as well as in keratinocyte proliferation. Therefore, inhibition of PKC in general might have a therapeutic benefit in psoriasis.

Alkaloids↗

PubMed1990

Evidence for a specific recognition site for tiflucarbine on calmodulin.

The putative antidepressant drug tiflucarbine (BAY P 4495) has previously been shown to inhibit calmodulin-dependent cyclic nucleotide phosphodiesterase competitively with respect to calmodulin. In order to determine whether this effect is mediated by a direct interaction with calmodulin, we measured the effects of radiolabelled triflucarbine in a direct ligand binding assay, using agarose-immobilized calmodulin. [3H]Tiflucarbine associated with low micromolar affinity with an apparently homogeneous class of recognition sites on calmodulin-agarose. No binding could be observed on calmodulin-deficient agarose. The effect was specific, saturable and reversible. Triflucarbine was the most potent calmodulin antagonist from a variety of structural analogues examined. The potencies of these derivatives to inhibit calmodulin-stimulated phosphodiesterase significantly correlated with their affinities towards the tiflucarbine binding site on calmodulin. No such correlation was evident when structurally unrelated reference compounds were tested. The association of tiflucarbine with calmodulin thus appears pharmacologically specific and selective and possibly contributes to the potent antidepressant activity of the drug.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1989

Non classical, multiple-site interaction of [3H]-prazosin with the alpha 1-adrenoceptor of intact BC3H1 cells.

1. In intact BC3H1 cells the EC50 of noradrenaline (NA) for the inositol phosphate response measured at 37 degrees C (EC50 = 193 nM) was much lower than its apparent dissociation constant (Ki37 degrees C = 83.211 microM) determined at this temperature by [3H]-prazosin binding. 2. After pretreatment of the cells with NA at 37 degrees C for 45 min, the time used in binding assays at this temperature, this difference between EC50 and Ki37 degrees C did not decrease significantly. An agonist-induced reduction in alpha 1-adrenoceptor affinity can therefore not explain the very high Ki37 degrees C value. 3. NA pretreatment at 37 degrees C decreased the number of [3H]-prazosin binding sites (assessed by whole cell binding at 2 degrees C) by only 49%; not by 100%, the value expected if agonist-induced receptor internalization were the origin of the very low Ki37 degrees C. 4. The EC50 of NA for the inositol phosphate response in the presence of 156 pM [3H]-prazosin was 1.841 microM but the IC50 of NA for the inhibition of [3H]-prazosin binding (126 pM) was 316 microM. As there is no alpha 1-adrenoceptor reserve in these cells we propose that at 37 degrees C [3H]-prazosin interacts, not only with the catecholamine recognition site (site 1) of the receptor, but also reacts weakly with another site from which it cannot be directly displaced by catecholamine-like substances (site 2).

Binding, Competitive↗

PubMed1988

New insights into maitotoxin action.

Maitotoxin (3 ng/mol) induced a massive uptake of 45Ca2+ into BC3H1 cells. This effect exhibits a lag phase of 3 min. Inositol diphosphate formation occurred concomittantly with the 45Ca2+ uptake but inositol monophosphate formation was found only after a 5-min delay following toxin addition. Maitotoxin-induced 45Ca2+ influxes could not be blocked by either 1 microM verapamil, 1 microM nifedipine or 1 mM La3+ but was blocked by Zn2+ (IC50 = 41 microM). In addition to inositol phosphate formation and 45Ca2+ uptake, maitotoxin stimulated a large uptake of Na+ and a great loss of K+ in BC3H1 cells. In the absence of Ca2+ (1 mM EGTA) none of the four maitotoxin effects could be detected. After restoration of Ca2+, the maitotoxin effects reappeared even when the toxin itself was no longer present. The divalent cation, Co2+ (1 mM), inhibited ion movements induced by maitotoxin and also digitonin (8.1 microM). The toxin action showed a very pronounced pH dependence. At low pH, maitotoxin was inactive. The dose-response curves for H+ ion inhibition of maitotoxin-induced Ca2+ uptake showed a shift to the right when determined in the absence of HCO3- and HCO3-/Cl- ions. It was concluded that the primary action of maitotoxin in BC3H1 cells was a pore-forming or channel-forming activity of a non-classical type. Some properties of maitotoxin resemble those of alpha-latrotoxin, others those of pore-forming agents such as melittin or alpha-toxin of Staphylococcus aureus.

Animals↗

PubMed1988

Neurotransmitter-induced inositol phosphate formation in neurons in primary culture.

Inositol-1,4,5-trisphosphate, produced in cells as a breakdown product of phosphatidylinositol-4,5-bisphosphate, induces, in many cell types, release of calcium from intracellular stores. In murine striatal neurons, differentiated in primary culture, carbachol, norepinephrine, glutamate, and neurotensin stimulate 3H-labeled inositol phosphate (3H-IP) production. The glutamate response was recently characterized as being mediated primarily by receptors of the quisqualate subtype. In the present study, we found that major differences exist between glutamate-stimulated 3H-IP formation and those stimulated by the other neuromediators. The maximal response to glutamate occurred before and during synaptogenesis and declined thereafter, whereas the maximal response to either carbachol or norepinephrine required complete neuronal differentiation. Although the glutamate response appears to be mediated exclusively by direct interaction with the neurotransmitter receptors, responses to carbachol, norepinephrine, and neurotensin were partially or completely blocked by tetrodotoxin.

Animals↗

PubMed1988

Complex interactions of agonists with alpha 1-adrenoceptors in intact cells.

1. The apparent Ki values of (-)-noradrenaline (NA), (+)- and (-)-adrenaline (Ad), phenylephrine and the mono-fluorinated NAs (in position 2, 5 or 6) for alpha 1-adrenoceptors of intact BC3H1 cells labelled with [3H]-prazosin were greatly dependent on the incubation temperature. 2. The EC50 values of these compounds for stimulation of the inositol phosphate (IP) accumulation at 37 degrees C were intermediate between their apparent dissociation constants at 2 degrees C (Ki2 degrees) and at 37 degrees C (Ki37 degrees). 3. The fact that an irreversible blockade of 46% +/- 6% (n = 3) of the [3H]-prazosin binding sites by phenoxybenzamine reduced the maximal IP-formation induced by NA by 57% +/- 5% (n = 3) shows that there is a direct coupling between alpha 1-adrenoceptors and phospholipase C in BC3H1 cells. 4. The Ki37 degrees s of all agonists tested were in the same range (0.1 to 1 mM) and showed no simple correlation with their EC50 values. 5. The Ki2 degrees values for all the agonist correlated linearly with their EC50 values but were about 20-100 times lower than the respective EC50 values (except for the partial agonist methoxamine). In order to explain this difference, we propose that the apparent high affinity in the cold could be due to an [3H]-prazosin-induced alteration of the active site of the alpha 1-adrenoceptor, increasing its apparent affinity for catecholamines.

Adrenergic alpha-Agonists↗

PubMed1987

Dual action of excitatory amino acids on the metabolism of inositol phosphates in striatal neurons.

Glutamate is able to stimulate inositol phosphate (IP) formation in striatal neurons in primary culture, mainly via an excitatory amino acid receptor of the quisqualate subtype. In the present study we show that carbachol (Carb)-(a cholinergic agonist), but not neurotensin or norepinephrine-induced IP production could be reduced by 40% when measured in the presence of Glu. The inhibition of the Carb response by Glu was dose dependent and reproduced by N-methyl-D-aspartate (NMDA). Quisqualate elicited an additive response with Carb. 2-Amino-5-phosphonovalerate (APV) completely reversed the NMDA-induced inhibition. APV had no significant effect on Glu- or kainate-induced inhibition. Therefore, striatal neurons contain at least three different excitatory amino acid receptors: a quisqualate receptor triggering the stimulation of IP metabolism, and an NMDA and a kainate receptor, both able to decrease the Carb-induced IP formation.

2-Amino-5-phosphonovalerate↗

PubMed1986

The potential antidepressant tiflucarbine down-regulates beta-adrenoceptors in rat brain.

Subchronic treatment of rats with the potential antidepressant tiflucarbine down-regulated the noradrenaline (NA) responses of the cAMP system in cerebral cortex. A concomitant 25% decrease in dihydroalprenolol binding sites in cerebral cortical membranes was observed. The effect was dose-dependent (ED50 = 6 mg/kg), required a 9 days' treatment period, and was reversible 5 days after discontinuation of treatment. Tiflucarbine treatment increased the specific activity of soluble calmodulin (CaM)-dependent phosphodiesterase in rat brain. Tiflucarbine bound to CaM and inhibited its interaction with the phosphodiesterase. Adrenergic denervation by 6-hydroxydopamine (6-OHDA) injection prevented both the beta-adrenoceptor down-regulation and the increase in specific activity of the phosphodiesterase. We suggest that a synergistic interaction between a presynaptic phosphodiesterase inhibition and the NA reuptake blockade was responsible for the down-regulation induced by tiflucarbine. The data are compatible with the reported antidepressant properties of this drug.

Adenylyl Cyclase Inhibitors↗

PubMed1986

Rolipram, a stereospecific inhibitor of calmodulin-independent phosphodiesterase, causes beta-adrenoceptor subsensitivity in rat cerebral cortex.

Prolonged pretreatment of rats with the atypical antidepressant rolipram attenuates noradrenaline (NA) sensitivity of the cerebral cortical cAMP generating system. The development of this down-regulation is time (7 d treatment required) and dose dependent (EC50 = 0.35 mg/kg). Density of beta-adrenoceptor as measured by (-)-3H-dihydroalprenolol [(-)-3H-DHA] binding is also reduced by rolipram pretreatment. The effect of rolipram is absolutely stereospecific for the (-)-enantiomer (ED50 = 0.18 mg/kg). In addition, only with this isomer, a reduction in daily weight gain was found compared to sham treated controls. Presynaptic denervation using intracerebroventricular (i.c.v.) injections of 6-hydroxydopamine (6-OHDA) prior to or during rolipram treatment did not completely block the effect of a rolipram treatment on down-regulation of cerebral cortical beta-adrenoceptors. The data favor a pre- and postsynaptic action of rolipram different from all other antidepressants studied so far in this experimental setting. Rolipram is known as inhibitor of brain phosphodiesterase. Using partially purified calmodulin-independent phosphodiesterase from brain it is shown that exclusively the (-)-enantiomer of rolipram inhibits phosphodiesterase with an IC50 of 1.25 mumol/l whereas the (+)-isomer possesses little potency. Since a marked stereospecificity for the (-)-isomer of rolipram was displayed in all pharmacological parameters tested so far with (+)- and (-)-rolipram, it is suggested that stereospecific and isozyme specific inhibition of cAMP-phosphodiesterase is, at least in part, related to the mechanism of action of the potential antidepressant drug rolipram and possibly of other antidepressants as well.

Adenylyl Cyclases↗

PubMed1985

Chronic thyroxine treatment of rats down-regulates the noradrenergic cyclic AMP generating system in cerebral cortex.

Treatment of Sprague-Dawley rats for 9 days with 15 micrograms/kg of tri-iodo-thyronine, 50 micrograms/kg of thyroxine (T4) or 500 micrograms/kg of thyrotropin-releasing hormone decreased the number of beta receptors in cerebral cortex as measured by dihydroalprenolol binding. The dissociation constants of dihydroalprenolol (3.5 nM) were not altered by the treatment. Only tri-iodo-thyronine and T4 administration resulted in a concomitant reduction of norepinephrine (NE) elicited cyclic AMP formation in cerebral cortical slices. This process required at least a 7-day treatment period and was dose-dependent. Application of 1 microgram of T4 per kg for 9 days significantly diminished the NE responsivity of the cyclic AMP synthesizing system. Dose-response curves with NE indicate a reduction of the maximal response after T4 treatment with no change in ED50. An almost additive interaction between the effects of T4 and a low dose (3 mg/kg) of the tricyclic antidepressant imipramine was observed. Striking differences in the response of the adrenoceptor coupled adenylate cyclase to a 9-day T4 treatment were found when different rat strains, i.e., Fischer F-344, Long-Evans, Wistar and Sprague-Dawley were used. The hyperthyroid state of the animals was ascertained by measurements of plasma levels of tri-iodo-thyronine and T4. Down-regulation of NE sensitive adenylate cyclase by T4 treatment required intact synaptic structures because denervation by i.c.v. injection of 6-hydroxydopamine abolished the effect of T4 treatment. This is indicative of a postsynaptic localization of the down-regulated cyclic AMP generating system. The data stress the importance of the neuroendocrine system for adrenoceptor regulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1983

Rapid Degradation of Isolated Lignins by Phanerochaete chrysosporium.

Phanerochaete chrysosporium degraded purified Kraft lignin, alkali-extracted and dioxane-extracted straw lignin, and lignosulfonates at a similar rate, producing small-molecular-weight ( approximately 1,000) soluble products which comprised 25 to 35% of the original lignins. At concentrations of 1 g of lignin liter, 90 to 100% of the acid-insoluble Kraft, alkali straw, and dioxane straw lignins were degraded by 1 g of fungal mycelium liter within an active ligninolytic period of 2 to 3 days. Cultures with biomass concentrations as low as 0.16 g liter could also completely degrade 1 g of lignin liter during an active period of 6 to 8 days. The absorbance at 280 nm of 2 g of lignosulfonate liter increased during the first 3 days of incubation and decreased to 35% of the original value during the next 7 days. The capacity of 1 g of cells to degrade alkali-extracted straw lignin under optimized conditions was estimated to be as high as 1.0 g day. This degradation occurred with a simultaneous glucose consumption rate of 1.0 g day. When glucose or cellular energy resources were depleted, lignin degradation ceased. The ability of P. chrysosporium to degrade the various lignins in a similar manner and at very low biomass concentrations indicates that the enzymes responsible for lignin degradation are nonspecific.

Journal Article↗