Differences in the blocking action of benzocaine and amines on batrachotoxin-modified sodium channels of the Ranvier node

Experiments by the voltage clamp method on Ranvier nodes of the frog nerve fiber showed that batrachotoxin sharply reduces the sensitivity of sodium channels to the blocking action of various tertiary (trimecaine, procaine, ajmaline, strychnine) and quaternary (QX-572, N-propylajmaline) amines but has no appreciable effect on blocking of sodium channels by neutral benzocaine. Inhibition of batrachotoxin-modified sodium currents by trimecaine is distinctly time-and potential-dependent in character. None of the amines tested gives rise to frequency-dependent (cumulative) blocking of the modified channels. Unblocking of these channels during rinsing of the node takes place much faster than unblocking of normal channels. The time course of recovery of the normal and modified currents after blocking by benzocaine is about the same. Relations between batrachotoxin "receptors" and the various blocking agents in the sodium channel are discussed.A. V. Vishnevskii Institute of Surgery, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 14, No. 6, pp. 636–643, November–December, 1982.     

Possible mechanism of long-term anti-arrhythmia and local anesthetic effect of quaternary ammonium compounds

In experiments with dialized neurons of L. stagnalis mollusc the recovery of Na-current (INa) after its depression by local anesthetic trimecaine and its quaternary derivative N-ethyltrimecaine (G-88) was studied. A full recovery of INa within tens of seconds after washing off trimecaine but not G-88 was observed. The half-time for vanishing of INa use-dependent depression by G-88 was 17 minutes, and there was no substantial vanishing of tonic INa block even after an hour of G-88 washing off. A hypothesis is advanced that the long recovery time of INa is one of the mechanisms providing long pharmacological action of quaternary antiarrhythmic and local anesthetic ammonium compounds.     

Transformation of LMTK- cells with purified class I genes. V. Antibody-induced structural modification of HLA class I molecules results in potentiation of the fixation of a second monoclonal antibody

A potentiation phenomenon was observed with HLA-A3 and CW3 transformed murine L cells between anti-HLA class I B10.6 (potentiated) and B10.8 (potentiating) monoclonal antibodies (m.Ab.). Further studies of this phenomenon with these transformed L cells indicated that: 1) no significant specific binding of B10.6 m.Ab. to HLA-A3 and CW3 transformed L cells could be demonstrated by conventional radioimmunoassay or cytofluorometric study in the absence of B10.8 m.Ab.; 2) potentiation of the fixation of B10.6 m.Ab. was induced by other anti-HLA class I m.Ab., which all reacted with the same cluster of antigenic determinants; 3) potentiation reflects an increased specific fixation of B10.6 m.Ab. to HLA class I molecules implicating its combining site; 4) potentiation was mediated by B10.8 Fab fragments. These results indicate that potentiation of the fixation of B10.6 m.Ab. to the HLA-A3 and CW3 molecules expressed by the transformed L cells reflects conformational changes of these molecules after interaction with B10.8 m.Ab.     

Role of surface phenomena in the mechanism of action of antiarrhythmic substances]

The surface activity of the antiaarhythmic drugs and their influence on the lipid-containing interphasics were studied. It was found that drug No. 7351 (diphenyl isopropyl acetic acid diethyl aminopropyl ester), fubromegan, metamysil, propranolol, quinidine, novocalinamide, xylocaine, and trimecaine possessed surface activity. There exists a symbate relationship between the surface and especially the interphasic activity and the antiarrhythmic effect. The most active antiarrhythmic drug No. 7351 increased the electric conductivity of the lecithine bilayer membrane much more than novocainamide.     

Decrease in maximal sodium permeability and slow sodium inactivation produced by trimecaine in the Ranvier node

The action of trimecaine on sodium permeability of the membrane was studied by the voltage clamp method in single nodes of Ranvier. Like procaine, trimecaine was shown to reduce maximal sodium permeability ( _Na) and to induce slow sodium inactivation. The slow inactivation arising in the presence of trimecaine was qualitatively very similar to that observed during the action of procaine or a high external potassium concentration. Dose-effect curves were obtained for _Na and slow inactivation and they showed that one molecule of trimecaine reacts with one sodium channel in the mechanism of both these effects. The local anesthetic has greater affinity for the receptor responsible for slow inactivation (equilibrium constants about 0.3·10^–3 M for _Na and 0.2·10^–14 M for slow inactivation.A. V. Vishnevskii Institute of Surgery, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 8, No. 4, pp. 418–425, July–August, 1976.     

Inhibition by sodium bromide of synaptic potentiation and calcium accumulation in the superior cervical ganglion of rat

Electrophysiological data show that sodium bromide (NaBr) inhibits the development of the post-tetanic potentiation in superior cervical ganglia of rat. NaBr treatment also prevented the accumulation of calcium in synaptic vesicles and dendritic vacuoles otherwise seen for some minutes after a tetanizing stimulation. These observations support the notion that the calcium ions entering into pre- and postsynaptic elements play an important role in the genesis of synaptic potentiation.     

Synthesis of (1S,2R)-1-phenyl-2-[(S)-1-aminopropyl]-N,N-diethylcyclopropanecarboxamide (PPDC) derivatives modified at the carbamoyl moiety as a new class of NMDA receptor antagonists

(1S,2R)-1-Phenyl-2-[(S)-1-aminopropyl]-N,N-diethylcyclopropanecarboxamide (PPDC, ), which is a conformationally restricted analogue of the antidepressant milnacipran [(+/-)-1], represents a new class of potent NMDA receptor antagonists. A series of PPDC analogues modified at the carbamoyl moiety were synthesized. Among these, (1S,2R)-1-phenyl-2-[(S)-1-aminopropyl]-N,N-dipropylcyclopropanecarboxamide (4d) was identified as the most potent NMDA receptor antagonist in this series and clearly reduced the MMDA receptor mediated potentiation of rat hippocampal slices, a model of long-term potentiation (LTP). The three-dimensional structure of 4d was also analyzed in detail to clarify the receptor-binding conformation.     

Plasminogen activation by tissue activator is accelerated in the presence of fibrin(ogen) cyanogen bromide fragment FCB-2

Fibrin, in contrast to fibrinogen, strongly accelerates the plasminogen activation by extrinsic activator (tissue-type plasminogen activator, t-PA). However, when fibrin and fibrinogen are digested with cyanogen bromide, both digests potentiate the t-PA-mediated plasminogen activation equally well. In this report, evidence is presented that this potentiating activity resides in CNBr fragment FCB-2 (= Ho1-DSK) and that a polymeric structure such as fibrin is not a prerequisite for the potentiation.     

Mechanism of local anesthesia: long-range orientation effects

The orientation interaction of a molecule of a local anesthetic with a biomembrane and cell-like liquid was studied, based on the model of adsorption of the anesthetic from a cell-like solution on the surface of a biomembrane for compounds of the trimecaine series. A statistically significant correlation equation was obtained, which relates the minimum blocking concentration to the projection of the dipole moment of the anesthetic on the plane X(1)0X2 of the principal axes of inertia. A model is prosed according to which the "anesthetic-biomembrane" interaction is most effective the molecule of the anesthetic rotates around the axis of the maximum moment of inertia.     

Andrographolide reduces cognitive impairment in young and mature AβPPswe/PS-1 mice

Alzheimer’s disease (AD) is a neurodegenerative disorder in which the amyloid-β (Aβ) oligomers are a key factor in synaptic impairment and in spatial memory decline associated with neuronal dysfunction. This impairment includes synaptic failure associated with the loss of synaptic proteins that contribute to AD progression. Interestingly, the use of natural compounds is an emergent conceptual strategy in the search for drugs with therapeutic potentials for treating neurodegenerative disorders. In the present study, we report that andrographolide (ANDRO), which is a labdane diterpene extracted from Andrographis paniculata, increases slope of field excitatory postsynaptic potentials (fEPSP) in the CA1 region of hippocampal slices and inhibits long-term depression (LTD), protecting the long-term potentiation (LTP) against the damage induced by Aβ oligomers in vitro, most likely by inhibiting glycogen synthase kinase-3β (GSK-3β). Additionally, ANDRO prevents changes in neuropathology in two different age groups (7- and 12-month-old mice) of an AβPPswe/PS-1 Alzheimer’s model. ANDRO reduces the Aβ levels, changing the ontogeny of amyloid plaques in hippocampi and cortices in 7-month-old mice, and reduces tau phosphorylation around the Aβ oligomeric species in both age groups. Additionally, we observed that ANDRO recovers spatial memory functions that correlate with protecting synaptic plasticity and synaptic proteins in two different age groups. Our results suggest that ANDRO could be used in a potential preventive therapy during AD progression.     

Similarity of behaviour of granule cells in hippocampus to that predicted by informon theory

In a “population” experiment on potentiation in hippocampus a large electrode in the perforant path (pp) stimulates granule cells which in turn excite basket cells; basket cells inhibit granule cells. A large electrode in the molecular layer among synapses from pp to granule cells records a “population EPSP”; if the electrode is placed in the granule cell body layer it records a “population spike” which is related to the discharge rate of granule cells. In the simulation of a granule/basket cell pair previously described the corresponding theoretical quantities are “postsynaptic state” and “internal state of the neurone” which determines its discharge rate.Experimental and theoretical waveforms in response to pp stimulation are compared; they agree reasonably well over a wide range of stimulus frequency and duration. The theory offers an explanation of some of the experimental waveforms.A prediction is made; some drugs increase and some drugs decrease the effectiveness of inhibitory synapses. A drug which increases the inhibitory effect of basket cells should, as a long term effect, increase the potentiation of the variable synapses from pp to granule cells and conversely, drugs which decrease inhibition should decrease potentiation.     

Vitamin K1 enhances sorafenib-induced growth inhibition and apoptosis of human malignant glioma cells by blocking the Raf/MEK/ERK pathway

ABSTRACT: BACKGROUND: The combined effects of anticancer drugs with nutritional factors against tumor cells have been reported previously. This study characterized the efficacy and possible mechanisms of the combination of sorafenib and vitamin K1 (VK1) on glioma cell lines. METHODS: We examined the effects of sorafenib, VK1 or their combination on the proliferation and apoptosis of human malignant glioma cell lines (BT325 and U251) by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, flow cytometry and 4',6-diamidino-2-phenylindole (DAPI) assay. The signaling pathway changes were detected by western blotting. RESULTS: Sorafenib, as a single agent, showed antitumor activity in a dose-dependent manner in glioma cells, but the effects were more pronounced when used in combination with VK1 treatment. Sorafenib in combination with VK1 treatment produced marked potentiation of growth inhibition and apoptosis, and reduced expression of phospho-mitogen-activated protein kinase kinase (MEK) and phospho-extracellular signal-regulated kinase (ERK). Furthermore, the expression levels of antiapoptotic proteins Bcl-2 and Mcl-1 were significantly reduced. CONCLUSIONS: Our findings indicated that VK1 enhanced the cytotoxicity effect of sorafenib through inhibiting the Raf/MEK/ERK signaling pathway in glioma cells, and suggested that sorafenib in combination with VK1 maybe a new therapeutic option for patients with gliomas.     

The effect of several nucleic acid binding drugs on the cleavage of d(GGAATTCC) and pBR 322 by the Eco RI restriction endonuclease

The endonucleolytic action of the EcoRI restriction enzyme on the double-stranded oligonucleotide d(GGAATTCC) and the supercoiled plasmid DNA pBR 233 is inhibited by actinomycin D, ethidium bromide, proflavin, distamycin A and netropsin. Half-maximal inhibition is observed at around 100 microM concentrations for the intercalating drugs, and around 0.1 to 1 microM concentrations for netropsin and distamycin A. The inhibitory activity of these drugs can be correlated with their affinity to the oligonucleotide and the plasmid DNA. Since at high concentrations of the drugs a complete inhibition is observed, it is concluded that the effect of the drugs on the stereochemistry of the EcoRI site is such that recognition is excluded.     

Desipramine selectively potentiates norepinephrine-elicited ERK1/2 activation through the α2A adrenergic receptor

The precise physiological effects of antidepressant drugs, and in particular their actions at non-monoamine transporter targets, are largely unknown. We have recently identified the tricyclic antidepressant drug desipramine (DMI) as a direct ligand at the α(2A) adrenergic receptor (AR) without itself driving heterotrimeric G protein/downstream effector activation [5]. In this study, we report our novel finding that DMI modulates α(2A)AR signaling in response to the endogenous agonist norepinephrine (NE). DMI acted as a signaling potentiator, selectively enhancing NE-induced α(2A)AR-mediated ERK1/2 MAPK signaling. This potentiation of ERK1/2 activation was observed as an increase in NE response sensitivity and a prolongation of the activation kinetics. DMI in a physiologically relevant ratio with NE effectively turned on ERK1/2 signaling that is lacking in response to physiological NE alone. Further, the DMI-induced ERK1/2 potentiation relied on heterotrimeric G(i/o) proteins and was arrestin-independent. This modulatory effect of DMI on NE signaling provides novel insight into the effects of this antidepressant drug on the noradrenergic system which it regulates, insight which enhances our understanding of the therapeutic mechanism for DMI.     

I–J loop involvement in the pharmacological profile of CLC-K channels expressed in Xenopus oocytes

CLC-K chloride channels and their subunit, barttin, are crucial for renal NaCl reabsorption and for inner ear endolymph production. Mutations in CLC-Kb and barttin cause Bartter syndrome. Here, we identified two adjacent residues, F256 and N257, that when mutated hugely alter in Xenopus oocytes CLC-Ka's biphasic response to niflumic acid, a drug belonging to the fenamate class, with F256A being potentiated 37-fold and N257A being potently blocked with a K_D ~ 1 μM. These residues are localized in the same extracellular I–J loop which harbors a regulatory Ca^2 + binding site. This loop thus can represent an ideal and CLC-K specific target for extracellular ligands able to modulate channel activity. Furthermore, we demonstrated the involvement of the barttin subunit in the NFA potentiation. Indeed the F256A mutation confers onto CLC-K1 a transient potentiation induced by NFA which is found only when CLC-K1/F256A is co-expressed with barttin. Thus, in addition to the role of barttin in targeting and gating, the subunit participates in the pharmacological modulation of CLC-K channels and thus represents a further target for potential drugs.     

Polymeric inhibitors of platelet aggregation. II. Copolymers of dipyridamole and related drugs with N-vinylpyrrolidone

A study has been made of the behaviour of the drugs dipyridamole and mopidamol (RA 233) attached to poly(N-vinylpyrrolidone). The inhibitory activities towards platelet aggregation induced by ADP or platelet activation factor (PAF) in sheep plasma have been examined and found to exceed the activities of the parent drugs, by factors up to 20. At the same time the abilities of the polymer-bound drugs in potentiating prostaglandin-type anti-aggregatory activities are much lower than those of the unattached drugs. The observations are explained in terms of polymer adsorption on to the platelet membranes, producing a high surface concentration of the drugs with consequent high inhibitory action. Intracellular action, such as the inhibition of phosphodiesterase, is reduced by the difficulty experienced by the polymeric drug in passing through the membrane, so that potentiation of prostaglandin activity, particularly against PAF, becomes small. A terpolymer containing units of dipyridamole and the prostaglandin analogue 5-(6-carboxyhexyl)-1-(3-cyclohexyl-3-hydroxypropyl)hydantoin (245C) shows a degree of ‘self-potentiation’.     

Effects of physostigmine and scopolamine on long-term potentiation of hippocampal population spikes in rats

To elucidate an involvement of the cholinergic system in the long-term potentiation phenomenon, effects of physostigmine and scopolamine on population spike and its long-term potentiation in the dentate granule cell layer of anesthetized rats and in the CA1 pyramidal cell layer of rat hippocampal slices were examined. In anesthetized rats, physostigmine (0.01 mg/kg, i.v.) enhanced at a late phase the long-term potentiation induced by tetanic stimulation (15 Hz, 15 s, 7.5 times the threshold for population spike) of the perforant path, while scopolamine (1.0 mg/kg) suppressed it at an early phase. The two drugs did not affect the population spike itself. The time course of the long-term potentiation under the treatment of physostigmine was similar to that induced by stronger tetanic stimulation (10 times the threshold). In hippocampal slices, physostigmine (10(-6)M) showed a tendency to enhance the long-term potentiation induced by tetanic stimulation (15 Hz, 15 s, 5 times the threshold) of the stratum radiatum, with an increase of the population spike itself. Scopolamine (10(-5)M) markedly suppressed the long-term potentiation with a decrease of the population spike itself. From these results, it is suggested that cholinergic modification by physostigmine or scopolamine affects the long-term potentiation phenomenon in the hippocampus under the in vivo and in vitro conditions.     

Inhibition of sodium currents in frog ranvier node treated with local anesthetics Role of slow sodium inactivation

Effects of different local anesthetics of sodium permeability were studied in single nerve fibres of frog by the method of voltage clamp. Inhibition of sodium current by externally applied tertiary anesthetics, procaine and trimecaine, was the sum of a potentially independent block (reduced $\text{P}{}_{\mathrm{rmNa}}$) and slow sodium inactivation with time constants ranging from tens to hundreds of ms depending on membrane potential (at room temperature). Externally applied uncharged benzocaine produced a potentially independent block only. According to dose-response curves both processes are one-to-one reactions. In the case of trimecaine equilibrium constant the reaction responsible for reduction of $\text{P}{}_{\mathrm{<mtext>Na</mtext>}}$ is about 0.3 mM, while that for slow inactivation is more than ten times less (0.02 mM). Increasing pH from 5.6 to 8.5 markedly accelerated the slow inactivation process at all potential values. Divalent cations Ca²⁺ and Ni²⁺ shifted the steady-state slow inactivation curve along the potential axis and simultaneously reduced slow inactivation at the saturation level. Permanently charged quaternary trimecaine was ineffective when applied externally. Internally applied tertiary anesthetics and quaternary trimecaine as well as externally applied quaternary derivative of lidocaine QX-572 produced a progressively irreversible block enhanced by depolarization and inhibition reversibly increased by repetitive short-term depolarization (frequency-dependent inhibition). Inhibition of sodium currents by repetitive stimulation observed also in the case of externally applied tertiary anesthetics is due mainly to slow inactivation. The data suggests the existence of several types of receptor sites through which local anesthetics exert their blocking action on sodium permeability.     

Cellular pharmacology of protein kinase Mζ (PKMζ) contrasts with its in vitro profile: implications for PKMζ as a mediator of memory

A number of recent studies have used pharmacological inhibitors to establish a role for protein kinase Mζ (PKMζ) in synaptic plasticity and memory. These studies use zeta inhibitory peptide (ZIP) and chelerythrine as inhibitors of PKMζ to block long term potentiation and memory; staurosporine is used as a negative control to show that a nonspecific kinase inhibitor does not block long term potentiation and memory. Here, we show that neither ZIP nor chelerythrine inhibits PKMζ in cultured cells or brain slices. In contrast, staurosporine does block PKMζ activity in cells and brain slices by inhibiting its upstream phosphoinositide-dependent kinase-1. These studies demonstrate that the effectiveness of drugs against purified PKMζ may not be indicative of their specificity in the more complex environment of the cell and suggest that PKMζ is unlikely to be the mediator of synaptic plasticity or memory.     

Bifunctional intercalation of antitumor antibiotics BBM-928A and echinomycin with deoxyribonucleic acid. Effects of intercalation on deoxyribonucleic acid degradative activity of bleomycin and phleomycin

The binding of peptide antitumor antibiotics, BBM-928A and echinomycin, to superhelical PM2 DNA and the effects of the resulting conformational changes of DNA on the DNA-degradative activity of two related antitumor antibiotics, bleomycin A2 and phleomycin D1, have been studied. The bifunctional intercalative mode of the DNA binding of BBM-928A concluded previously from viscometric and fluorometric studies has been confirmed by gel electrophoretic analysis. Under the employed electrophoretic conditions, DNA-bound BBM-928A showed little dissociation whereas echinomycin and ethidium bromide showed partial and nearly complete dissociation, respectively. BBM-928A induced neither single-strand nor double-strand breaks in DNA. Competitive binding studies by fluorescence changes suggested that binding sites on DNA molecules for BBM-928A (or echinomycin) may differ from those for ethidium bromide, since binding to DNA by the two drugs was not competitive even at saturating concentrations. The lack of such a competition between the two drugs is not consistent with the neighbor-exclusion principle. The DNA-degradative activity of both bleomycin A2 and phleomycin D1 increased with the removal of the negative superhelicity of DNA by the BBM-928A intercalation and decreased with the formation of positive superhelical turns induced by high concentrations of BBM-928A. Thus the degradative activity of both bleomycin A2 and phleomycin D1 is sensitive in a similar manner to the degree of superhelicity rather than the double helicity of DNA, although there are differences between these two drugs in interaction with DNA.