Novel Organoruthenium(II) Complex C1 Selectively Inhibits Butyrylcholinesterase without Side Effects on Neuromuscular Transmission.

Enzyme butyrylcholinesterase (BChE) shows increased activity in some brain regions after progression of Alzheimer's disease and is therefore one of the therapeutic targets for symptomatic treatment of this neurodegenerative disorder. The organoruthenium(II) complex [(η6-p-cymene)Ru(II)(1-hydroxy-3-methoxypyridine-2(1H)-thionato)pta]PF6 (C1) was designed based on the results of our previous structure-activity studies. Inhibitory activity toward cholinesterase enzymes shows that this complex selectively, competitively, and reversibly inhibits horse serum BChE (hsBChE) with an IC50 value of 2.88 µM. When tested at supra-pharmacological concentrations (30, 60, 90, and 120 µM), C1 had no significant effect on the maximal amplitude of nerve-evoked and directly elicited single-twitch and tetanic contractions. At the highest tested concentration (120 µM), C1 had no effect on resting membrane potential, but significantly decreased the amplitude of miniature end-plate potentials (MEPP) without reducing their frequency. The same concentration of C1 had no effect on the amplitude of end-plate potentials (EPP), however it shortened the half-decay time of MEPPs and EPPs. The decrease in the amplitude of MEPPs and shortening of the half-decay time of MEPPs and EPPs suggest a possible weak inhibitory effect on muscle-type nicotinic acetylcholine receptors (nAChR). These combined results show that, when applied at supra-pharmacological concentrations up to 120 µM, C1 does not importantly affect the physiology of neuromuscular transmission and skeletal muscle contraction.

The first Kunitz-type proteins from a viperid venom that potentiate neuromuscular transmission.

Kunitz-type proteins that interfere with neuronal transmission have been thus far exclusively detected in venoms of elapid snakes. Here, we report for the first time that such proteins are also present in the venom of a viperid snake. From the venom of the nose-horned viper (Vipera ammodytes ammodytes; Vaa), we isolated Kunitz-type chymotrypsin inhibitors (VaaChi) and demonstrated that these molecules also significantly increase the amplitudes of an indirectly evoked simple muscle contraction of the mouse hemidiaphragm, the end-plate potential and the miniature end-plate potential. By facilitating neuromuscular transmission, these proteins resemble structurally homologous dendrotoxins from mamba (Dendroaspis spp.) venoms, which are blockers of voltage-dependent K+ channels at the presynaptic site of the neuromuscular junction. What is the mechanism behind facilitation of neuromuscular transmission by VaaChi has not been established yet, however, blocking of K+ channels does not seem to be the most probable option.

Structural and functional characterization of an organometallic ruthenium complex as a potential myorelaxant drug.

In addition to antibacterial and antitumor effects, synthetic ruthenium complexes have been reported to inhibit several medicinally important enzymes, including acetylcholinesterase (AChE). They may also interact with muscle-type nicotinic acetylcholine receptors (nAChRs) and thus affect the neuromuscular transmission and muscle function. In the present study, the effects of the organometallic ruthenium complex of 5-nitro-1,10-phenanthroline (nitrophen) were evaluated on these systems. The organoruthenium-nitrophen complex [(η6-p-cymene)Ru(nitrophen)Cl]Cl; C22H21Cl2N3O2Ru (C1-Cl) was synthesized, structurally characterized and evaluated in vitro for its inhibitory activity against electric eel acetylcholinesterase (eeAChE), human recombinant acetylcholinesterase (hrAChE), horse serum butyrylcholinesterase (hsBChE) and horse liver glutathione-S-transferase. The physiological effects of C1-Cl were then studied on isolated mouse phrenic nerve-hemidiaphragm muscle preparations, by means of single twitch measurements and electrophysiological recordings. The compound C1-Cl acted as a competitive inhibitor of eeAChE, hrAChE and hsBChE with concentrations producing 50 % inhibition (IC50) of enzyme activity ranging from 16 to 26 µM. Moreover, C1-Cl inhibited the nerve-evoked isometric muscle contraction (IC50 = 19.44 µM), without affecting the directly-evoked muscle single twitch up to 40 µM. The blocking effect of C1-Cl was rapid and almost completely reversed by neostigmine, a reversible cholinesterase inhibitor. The endplate potentials were also inhibited by C1-Cl in a concentration-dependent manner (IC50 = 7.6 µM) without any significant change in the resting membrane potential of muscle fibers up to 40 µM. Finally, C1-Cl (5-40 µM) decreased (i) the amplitude of miniature endplate potentials until a complete block by concentrations higher than 25 µM and (ii) their frequency at 10 µM or higher concentrations. The compound C1-Cl reversibly blocked the neuromuscular transmission in vitro by a non-depolarizing mechanism and mainly through an action on postsynaptic nAChRs. The compound C1-Cl may be therefore interesting for further preclinical testing as a new competitive neuromuscular blocking, and thus myorelaxant, drug.

Organoruthenium Prodrugs as a New Class of Cholinesterase and Glutathione-S-Transferase Inhibitors.

A small library of 17 organoruthenium compounds with the general formula [RuII (fcl)(chel)(L)]n+ (in which fcl=face capping ligand, chel=chelating bidentate ligand, and L=monodentate ligand) were screened for inhibitory activity against cholinesterases and glutathione-S-transferases of human and animal origins. Compounds were selected to include different chelating ligands (i.e., N,N-, N,O-, O,O-, S,O-) and monodentate ligands that can modulate the aquation rate of the metal species. Compounds with a labile ruthenium chloride bond that provided rapid aquation were found to inhibit both sets of enzymes in reversible competitive modes and at pharmaceutically relevant concentrations. When applied at concentrations that completely abolish the activity of human acetylcholinesterase, the lead compound [(η6 -p-cymene)Ru(pyrithionato)Cl] (C1 a) showed no undesirable physiological responses on the neuromuscular system. Finally, C1 a was not cytotoxic against non-transformed cells at pharmaceutically relevant concentrations.

Analysis of compound action potentials elicited with specific current stimulating pulses in an isolated rat sciatic nerve.

The ability to selectively stimulate Aα, Aß-fibers and Aδ-fibers in an isolated rat sciatic nerve (SNR) was assessed. The stimulus used was a current, biphasic pulse with a quasitrapezoidal cathodic phase and rectangular anodic phase where parameters were systematically varied: intensity of the cathodic phase (ic); width of the cathodic phase (tc); width of the cathodic exponential decay (texp) and time constant of the exponential decay (τexp). A SNR was stimulated using a pair of hook electrodes while conduction velocity (CV) and compound action potentials (CAP) were measured at two sites along the SNR using another two pairs of electrodes. Results showed that the highest CAP1 (8.5-9 mV), shall be expected when parameters of the stimulus were within the following range: ic=3.8-4 mA, tc=350-400 µs and texp=330-440 µs. Results also showed that with ascending tc and texp, CV of the corresponding superficial region of the SNR was reduced in both, conduction velocity of CAP1 and conduction velocity of CAP2. It was concluded that action potentials (APs) were activated in the Aß-fibers and Aδ-fibers along with a slight AP inhibition in the Aß-fibers. The obtained results, could serve as a tool for developing multi-electrode systems that potentially enable fiber-type selective stimulation of nerve fibers.

Synthetic analogs of stryphnusin isolated from the marine sponge Stryphnus fortis inhibit acetylcholinesterase with no effect on muscle function or neuromuscular transmission.

The marine secondary metabolite stryphnusin (1) was isolated from the boreal sponge Stryphnus fortis, collected off the Norwegian coast. Given its resemblance to other natural acetylcholinesterase antagonists, it was evaluated against electric eel acetylcholinesterase and displayed inhibitory activity. A library of twelve synthetic phenethylamine analogs, 2a-7a and 2b-7b, containing tertiary and quaternary amines respectively were synthesized to investigate the individual structural contributions to the activity. Compound 7b was the strongest competitive inhibitor of both acetylcholinesterase and butyrylcholinesterase with IC50 values of 57 and 20 µM, respectively. This inhibitory activity is one order of magnitude higher than the positive control physostigmine, and is comparable with several other marine acetylcholinesterase inhibitors. The physiological effect of compound 7b on muscle function and neuromuscular transmission was studied and revealed a selective mode of action at the investigated concentration. This data is of importance as the interference of therapeutic acetylcholinesterase inhibitors with neuromuscular transmission can be problematic and lead to unwanted side effects. The current findings also provide additional insights into the structure-activity relationship of both natural and synthetic acetylcholinesterase inhibitors.

Venomics of Vipera berus berus to explain differences in pathology elicited by Vipera ammodytes ammodytes envenomation: Therapeutic implications.

UNLABELLED: Vipera berus berus (Vbb) is the most widely distributed and Vipera ammodytes ammodytes (Vaa) the most venomous viper in Europe. In particular areas of the Old continent their toxic bites constitute a considerable public health problem. To make the current envenomation therapy more effective we have analysed the proteome of Vbb venom and compared it with that of Vaa. We found the proteome of Vbb to be much less complex and to contain smaller levels of particularly snaclecs and sPLA2s. Snaclecs are probably responsible for thrombocytopenia. The neurotoxic sPLA2s, ammodytoxins, are responsible for the most specific feature of the Vaa venom poisoning - induction of signs of neurotoxicity in patients. These molecules were not found in Vbb venom. Both venoms induce haemorrhage and coagulopathy in man. As Vaa and Vbb venoms possess homologous P-III snake venom metalloproteinases, the main haemorrhagic factors, the severity of the haemorrhage is dictated by concentration and specific activity of these molecules. The much greater anticoagulant effect of Vaa venom than that of Vbb venom lies in its higher extrinsic pathway coagulation factor-proteolysing activity and content of ammodytoxins which block the prothrombinase complex formation. BIOLOGICAL SIGNIFICANCE: Envenomations by venomous snakes constitute a considerable public health problem worldwide, and also in Europe. In the submitted work we analysed the venom proteome of Vipera berus berus (Vbb), the most widely distributed venomous snake in Europe and compared it with the venom proteome of the most venomous viper in Europe, Vipera ammodytes ammodytes (Vaa). We have offered a possible explanation, at the molecular level, for the differences in clinical pictures inflicted by the Vbb and Vaa venoms. We have provided an explanation for the effectiveness of treatment of Vbb envenomation by Vaa antiserum and explained why full protection of Vaa venom poisoning by Vbb antiserum should not be always expected, especially not in cases of severe poisoning. The latter makes a strong case for Vaa antiserum production as we are faced with its shortage due to ceasing of production of two most frequently used products.

Effect of the ostreolysin A/pleurotolysin B pore-forming complex on intracellular Ca2+ activity in the vascular smooth muscle cell line A10.

Ostreolysin A/pleurotolysin B (OlyA/PlyB) is a binary pore-forming protein complex that produces a rapid cardiorespiratory arrest. Increased tonus of the coronary vascular wall produced by OlyA/PlyB may lead to ischemia, arrhythmias, the hypoxic injury of cardiomyocytes and cardiotoxicity. We evaluated the effects of OlyA/PlyB in cultured vascular smooth muscle A10 cells. Fluorometric measurements using the Ca(2+) indicator Fluo-4 AM and Fura-2 AM revealed that nanomolar concentrations of OlyA/PlyB increased the intracellular Ca(2+) activity [Ca(2+)]i in A10 cells. This effect was absent in a Ca(2+)-free medium, indicating that OlyA/PlyB-induced [Ca(2+)]i increase was dependent on Ca(2+) influx into cells. The increase in [Ca(2+)]i by OlyA/PlyB was partially prevented by: i) the calcium channel blockers verapamil and La(3+), ii) the inhibitor of the sodium-calcium exchanger (NCX) benzamil, and iii) the iso-osmotic replacement of NaCl by sucrose. The pre-treatment of cells with the Ca(2+)-ATPase inhibitor thapsigargin reduced the [Ca(2+)]i increase evoked by OlyA/PlyB, whereas the plasma membrane depolarization with high K(+) in the medium did not prevent OlyA/PlyB-induced [Ca(2+)]i. In summary, our data could suggest that the OlyA/PlyB-induced increase in [Ca(2+)]i is due to an influx of Ca(2+) through a variety of co-existing plasma membrane Ca(2+)-permeable channels, Ca(2+) entry through non-selective ion permeable pores formed de novo by OlyA/PlyB in the plasma membrane and calcium-induced intracellular Ca(2+) release, altogether leading to disturbed Ca(2+) homeostasis in A10 cells.

Retrospective study of bull semen quality - possible correlation with pesticide use?

Decline in semen quality in humans and increased incidence of male reproductive problems could be caused by different factors, including pesticides that could mimic or block the action of endogenous hormones. If the decline in semen quality is real, and environmental chemicals are at least partially responsible for this decline, similar changes should be observed in animals that live in close connection with humans and are exposed to similar levels of pollutants. In the present study, the semen quality of bulls in the last 30 years was examined with respect to the year of birth. Furthermore, semen quality results were correlated to the total pesticide use in a limited geographical area. The results indicate a notable decrease in both ejaculate volume and total number of spermatozoa in ejaculates of bulls born in the late 1970s, while after that (until 2006) there was no obvious downward or upward trend either in ejaculate volume or in the total number of sperm cells. The amount of pesticides released into the environment increased about twofold in the given period, and linear regression analysis revealed a strong and statistically significant correlation between the amount of pesticides used and the total number of spermatozoa in the ejaculate.