Bulky Adducts in Clustered DNA Lesions: Causes of Resistance to the NER System.

The nucleotide excision repair (NER) system removes a wide range of bulky DNA lesions that cause significant distortions of the regular double helix structure. These lesions, mainly bulky covalent DNA adducts, are induced by ultraviolet and ionizing radiation or the interaction between exogenous/endogenous chemically active substances and nitrogenous DNA bases. As the number of DNA lesions increases, e.g., due to intensive chemotherapy and combination therapy of various diseases or DNA repair impairment, clustered lesions containing bulky adducts may occur. Clustered lesions are two or more lesions located within one or two turns of the DNA helix. Despite the fact that repair of single DNA lesions by the NER system in eukaryotic cells has been studied quite thoroughly, the repair mechanism of these lesions in clusters remains obscure. Identification of the structural features of the DNA regions containing irreparable clustered lesions is of considerable interest, in particular due to a relationship between the efficiency of some antitumor drugs and the activity of cellular repair systems. In this review, we analyzed data on the induction of clustered lesions containing bulky adducts, the potential biological significance of these lesions, and methods for quantification of DNA lesions and considered the causes for the inhibition of NER-catalyzed excision of clustered bulky lesions.

Recognition and removal of clustered DNA lesions via nucleotide excision repair.

Clustered damage of DNA consists of two or more lesions located within one or two turns of the DNA helix. Clusters consisting of lesions of various structures can arise under the influence of strong damaging factors, especially if the cells have a compromised repair status. In this work, we analyzed how the presence of an analog of the apurinic/apyrimidinic site - a non-nucleoside residue consisting of diethylene glycol phosphodiester (DEG) - affects the recognition and removal of a bulky lesion (a non-nucleoside site of the modified DNA strand containing a fluorescein residue, nFlu) from DNA by a mammalian nucleotide excision repair system. Here we demonstrated that the efficiency of nFlu removal decreases in the presence of DEG in the complementary strand and is completely suppressed when the DEG is located opposite the nFlu. By contrast, protein factor XPC-RAD23B, which initiates global genomic nucleotide excision repair, has higher affinity for DNA containing clustered damage as compared to DNA containing a single bulky lesion; the affinity of XPC strengthens as the positions of DEG and nFlu become closer. The changes in the double-stranded DNA's geometry caused by the presence of clustered damage were also assessed. The obtained experimental data together with the results of molecular dynamics simulations make it possible to get insight into the structural features of DNA containing clustered lesions that determine the efficiency of repair. Speaking more broadly, this study should help to understand the probable fate of bulky adduct-containing clusters of various topologies in the mammalian cell.

Increased non-quantal release of acetylcholine after inhibition of endocytosis by methyl-ß-cyclodextrin: the role of vesicular acetylcholine transporter.

We investigated the role of the vesicular acetylcholine transporter in the mechanism of non-quantal (non-vesicular) secretion of neurotransmitter in the neuromuscular synapse of the rat diaphragm muscle. Non-quantal secretion was estimated electrophysiologically by the amplitude of end-plate hyperpolarization after inhibition of cholinesterase and nicotinic receptors (H-effect) or measured by the optical detection of acetylcholine in the bathing solution. It was shown that 1 mM methyl-ß-cyclodextrin (MCD) reduced both endocytosis and, to much lesser extent, exocytosis of synaptic vesicles (SV) thereby increasing non-quantal secretion of acetylcholine with a concurrent decrease in axoplasm pH. During high-frequency stimulation of the motor nerve, that substantially increases vesicles exocytosis, the non-quantal secretion was further enhanced if the endocytosis of SV was blocked by MCD. In contrast, non-quantal secretion of acetylcholine did not increase when the MCD-treated neuromuscular preparations were superfused with either vesamicol, an inhibitor of vesicular transporter of acetylcholine, or sodium propionate, which decreases intracellular pH. These results suggest that the proton-dependent, vesamicol-sensitive vesicular transporters of acetylcholine, which become inserted into the presynaptic membrane during SV exocytosis and removed during endocytotic recycling of SV, play the major role in the process of non-quantal secretion of neurotransmitter.

Interaction of glutamate- and adenosine-induced decrease of acetylcholine quantal release at frog neuromuscular junction.

In a frog neuromuscular preparation of m. sartorius, glutamate had a reversible dose-dependent inhibitory effect on both spontaneous miniature endplate potentials (MEPP) and nerve stimulation-evoked endplate potentials (EPP). The effect of glutamate on MEPP and EPP is caused by the activation of metabotropic glutamate receptors, as it was eliminated by MCPG, an inhibitor of group I metabotropic glutamate receptors. The depression of evoked EPP, but not MEPP frequency was removed by inhibiting the NO production in the muscle by L-NAME and by ODQ that inhibits the soluble NO-sensitive guanylyl cyclase. The glutamate-induced depression of the frequency of spontaneous MEPP is apparently not caused by the stimulation of the NO cascade. The particular glutamate-stimulated NO cascade affecting the evoked EPP can be down-regulated also by adenosine receptors, as the glutamate and adenosine actions are not additive and application of adenosine partially prevents the further decrease of quantal content by glutamate. On the other hand, there is no obvious interaction between the glutamate-mediated inhibition of EPP and inhibitory pathways triggered by carbacholine and ATP. The effect of glutamate on the evoked EPP release might be due to NO-mediated modulation (phosphorylation) of the voltage-dependent Ca2+ channels at the presynaptic release zone that are necessary for evoked quantal release and open during EPP production.

Adenosine triphosphoric acid as a factor of nervous regulation of Na+/K+/2Cl- cotransport in rat skeletal muscle fibers.

Exogenous adenosine triphosphoric acid produces a biphasic effect on the resting membrane potential of muscle fibers in rat diaphragm. Depolarization of the sarcolemma observed 10 min after application of adenosine triphosphoric acid results from activation of Na(+)/K(+)/2Cl(-) cotransport. The increase in chloride cotransport is related to activation of postsynaptic P2Y receptors and protein kinase C. Repolarization of the membrane develops 40 min after treatment with adenosine triphosphoric acid and after 50 min the resting membrane potential almost returns the control level. This increase in the resting membrane potential of the sarcolemma is probably associated with activation of the Na(+)/K(+) pump and increase in membrane permeability for chlorine ions in response to long-term activity of Cl(-) cotransport. Thus, adenosine triphosphoric acid co-secreted with acetylcholine in the neuromuscular synapse probably plays a role in the regulation resting membrane potential and cell volume of muscle fibers.

Effect of dipeptide N-acetylaspartylglutamate on denervation-induced changes in the volume of rat skeletal muscle fibers.

N-acetylaspartylglutamate prevents the denervation-induced increase in the volume of muscle fibers in rat diaphragm, the phenomenon being more pronounced for the hydrolysable isomer. The effect of dipeptide manifested against the background of blockade of metabotropic glutamate receptors. It was hypothesized that N-acetylaspartylglutamate is involved in the regulation of the volume of skeletal muscle fibers via activation of ionotropic receptors by both dipeptide and glutamate molecules.

Effect of oxotremorine on resting membrane potential and cell volume in skeletal muscle fibers in rats after in vivo blockade of NO-synthase.

Denervation of rat phrenic muscle or block of NO-synthase in vivo increased the cross-section area of muscle fibers and decreased membrane resting potential. Oxotremorine prevented the development of denervation-induced or denervation-like (i.e. induced by NO-synthase blockade) membrane depolarization and increase of the cross-sectional area of muscle fibers. Pirenzepine abolished the effects of oxotremorine. It was concluded that non-quantal acetylcholine can be involved in the regulation of skeletal muscle fiber volume via activation of M1 muscarinic receptors followed by NO synthesis.

[Pharmacological parameters of muscarinic cholinoreceptors in skeletal muscles]. / Farmakologicheskie osobennosti muskarinovykh cholinoretseptorov skeletnykh myshts.

It has been shown that bath application of muscarine delayed the early post-denervation depolarization in the muscle fibers incubated for 3 h in culture medium. The greatest reduction of the post-devervation depolarization was observed with 50 nmol/l muscarine. Atropine, a muscarinic antagonist, clozapine, a specific inhibitor of M1/M5-cholinergic receptors, and nitrocaramiphen, a M1-antagonist, completely removed the hyperpolarizing effect of muscarine. 4-DAMP, a specific inhibitor of M3-cholinergic receptors, himbacine, an antagonist of M2-cholinergic receptors, and tropicamide, a specific inhibitor of M2/M4-cholinergic receptors, failed to prevent the effect of muscarine. A M1/M2 muscarine agonists propargyl and but-2-ynyl esters of arecaidine had apparent muscarine-like effect. Nitrocaramiphen, and not himbacine, prevented the hyperpolarizing effect of these cholinomimetics. It is concluded that muscarine and esters of arecaidine delay the development of early postdenervation depolarization in M1-cholinergic receptors of skeletal muscle.

[Effect of glutamate on membrane potential and volume of the skeletal muscle fibers in rats following NO-synthase inhibition in vivo]. / Vliianie glutamata na membrannyi potentsial i ob''em skeletnykh myshechnykh volokon krysy posle blokady NO-sintazy in vivo.

Cross-sectional area (CSA) of muscle fibers incubated in culture medium 199 for 3 hours dramatically increases, whereas resting membrane potential (RMP) decreases compared to "freshly-isolated" muscles. Both glutamate and sodium nitroprusside prevent these changes. MK-801, a specific inhibitor of NMDA-receptors, eliminates protective effects of glutamate on both CSA and RMP. NO-synthase inhibition in vivo promotes an increase of initial CSA and decrease of mean RMP. Under these conditions, effects of glutamate and sodium nitroprusside on CSA and RMP of denervated muscles are less obvious. It has been concluded that synaptic glutamate is able to participate in regulation of RMP and cell volume in muscle fibers through the activation of postsynaptic NMDA-receptors and muscle NO-synthase.

The glutamate and carbachol effects on the early post-denervation depolarization in rat diaphragm are directed towards furosemide-sensitive chloride transport.

The membrane potentials of denervated muscle fibres of the rat diaphragm kept in a tissue culture medium are depolarized by about 8-10 mV (10-12%) within 3 h after denervation. This early post-denervation depolarization (EPD) is substantially reduced (2-3 mV) when muscle strips are bathed with 1 mM L-glutamate (GLU) which is found in motor nerve endings, or with 5 x 10(-8) M carbachol (CCh), which mimics the effect of nonquantally released acetylcholine (ACh). The hyperpolarizing effects of GLU and CCh on EPD are not influenced by ouabain, an active sodium transport inhibitor, but are absent when Cl- transport is augmented by increased osmolarity (500 mosmol/l) produced by addition of sucrose or NaCl. The EPD and the effect of hyperosmolarity are effectively prevented by the Cl- transport inhibitor furosemide (1 x 10(-4) M) or by a chloride-free bathing medium. It is suggested that the post-denervation cessation of nonquantal ACh release, and probably also GLU release, from nerve endings leads to the activation of the furosemide-sensitive Cl- transport in the sarcolemma, which is responsible for the early post-denervation depolarization.

Carnosine and other imidazole-containing compounds enhance the postdenervation depolarization of the rat diaphragm fibres.

In the presence of carnosine, anserine, histidine, imidazole and 7-nitro indazole, the early postdenervation depolarization of muscle of about 8 mV was significantly increased by 2.15-4.8 mV. The presence of the imidazole ring in the molecule is apparently necessary for this effect. These compounds also eliminated an NO-mediated protective effect of L-glutamate and carbachol on the depolarization of membrane potential. The presence of imidazole, 7-nitro indazole, carnosine and anserine did not significantly change the effect of an external NO donor, sodium nitroprusside. The structural and functional similarity between imidazole derivatives and the known NO synthase inhibitor, 7-nitro indazole suggests that imidazole, carnosine and anserine might act by inhibiting NO production which is stimulated by glutamate and carbachol.

The effect of glutamate and inhibitors of NMDA receptors on postdenervation decrease of membrane potential in rat diaphragm.

The early postdenervation depolarization of rat diaphragm muscle fibers (8-10 mV within 3 h in vitro) is substantially smaller (3 mV) when muscles are bathed with 1 x 10(-3) M L-glutamate (Glu) or 1 x 10(-3) M N-methyl-D-aspartate (NMDA). The effects of Glu and NMDA are inhibited in a dose-dependent manner by competitive inhibitor 2-amino-5-phosphonovaleric acid (APV) with Ki 6.3 x 10(-4) M, by 2 x 10(-7) M MK-801, which acts as an open channel inhibitor, by 2-3 x 10(-4) Zn2+, which reacts with surface-located sites of the NMDA subtype of the glutamate receptor, and also by glycine-free solutions and 7-Cl-kynurenic acid, which inhibits the glycine binding sites on NMDA receptors. It follows that the effect of glutamate on early post-denervation depolarization is mediated by the NMDA subtype of glutamate receptor with similar pharmacological properties to those found in neurons. The only exception found was the glutamate-like action of 1 x 10(-7) M MK-801, which partially prevented the early postdenervation depolarization when present in the muscle bath during the first 3 h after nerve section.

Acetylcholine and carbachol prevent muscle depolarization in denervated rat diaphragm.

Muscle fibres of the rat diaphragm kept in a tissue culture medium became depolarized by 8-10 mV within 3 h after denervation. In the presence of carbachol (CB; 5 x 10(-8) M), and acetylcholine (ACh; 5 x 10(-8) M, the post-denervation depolarization was reduced. Both drugs were used in concentrations which mimicked the effect of non-quantal release of ACh. (+)Tubocurarine (TC) and ouabain did not prevent the protective action of CB, indicating that this effect is not mediated through ACh nicotinic receptors or the electrogenic Na+, K+ pump. Addition of Mg2+, verapamil, diltiazem, nifedipine and Cd2+ in concentrations which block Ca2+ entry virtually inhibited the effect of both cholinomimetics. L-Nitroarginine methylester (NAME), an inhibitor of NO synthase, and haemoglobin, an extracellular scavenger of the NO radical, completely eliminated the protective effect of CB on post-denervation depolarization. The retrograde action of NO produced by cholinomimetics on nerve terminals is postulated.

Nitroprusside decreases the early post-denervation depolarization of diaphragm muscle fibres of the rat.

The application of sodium nitroprusside, which degrades to nitric oxide (NO) in solution, inhibits early post-denervation depolarization of isolated rat diaphragm fibres. The observation that "old' solutions of sodium nitroprusside (that have been allowed to decompose) are without effect and that haemoglobin, oxadiazolo quinoxalinone (ODQ) and methylene blue can antagonize the inhibition normally produced by sodium nitroprusside suggests that the inhibitory effects of sodium nitroprusside on early post-denervation depolarization are mediated by NO and guanylyl cyclase. This is in accord with our recent observations with NO synthase activation and inhibition in the diaphragm.