[ROLE PHOSPHOINOSITID SIGNALING PATHWAY IN OPIOIDS CONTROL OF P2X3 RECEPTORS IN THE PRIMARY SENSORY NEURONS].

Homomeric P2X3 receptors expressed in primary nociceptive neurons are crucial elements in the pain signal generation. In turn, opioid system regulates the intensity of this signal in both CNS and PNS. Here we describe the effects of opioids on P2X3 receptors in DRG neurons studied by using patch clamp technique. Activation of G-protein coupled opioid receptors by endogenous opioid Leu-enkephalin (Leu), resulted in the two opposite effects on P2X3 receptor-mediated currents (P2X3 currents). In particular, application of 1 µM Leu lead to the complete inhibition of P2X3 currents. However, after pretreatment of the neurons with a Gi/o-protein inhibitor pertussis toxin (PT), the same concentration of Leu caused facilitation of P2X3 currents. PLC inhibitor U-73122 at concentration of 1 µM completely eliminated both facilitating and inhibitory effects of Leu on P2X3 currents. Thus, opioid receptor agonists cause two oppositely directed effects on P2X3 receptors in DRG neurons of rats and both of them are mediated through PLC signaling pathway. Our results point to a possible molecular basis of the mechanism for the well-known transition inhibitory action of opioids (analgesia) to facilitating (hyperalgesia).

Extracellular cAMP inhibits P2X receptors in rat sensory neurones through G protein-mediated mechanism.

AIM: To identify the mechanisms of P2X(3) receptor inhibition by extracellular cyclic adenosine monophosphate (cAMP) in rat dorsal root ganglion (DRG) neurones. METHODS: Whole-cell currents were measured in cultured DRG neurones using the combination of voltage and concentration clamp. RESULTS: We have found that extracellular cAMP inhibits P2X(3)-mediated currents in a concentration- and use-dependent manner. The P2X(3) currents, activated by ATP applied every 4 min, were inhibited by 55% in the presence of 10 microm cAMP and by 81% in the presence of 30 microm cAMP. At 8 min interval between ATP applications the same concentration of cAMP did not alter the currents. Addition of 0.5 mm of guanosine 5'-O-(2-thiodiphosphate) to intracellular solution blocked the inhibitory action of cAMP. The inhibitory effects of cAMP were not mimicked by extracellular application of 30 mum adenosine. CONCLUSIONS: In this paper, we demonstrate, for the first time, that extracellular application of cAMP to rat sensory neurones inhibits P2X(3) receptors via a G protein-coupled mechanism in a use-dependent manner, thus indicating the neuronal expression of specific plasmalemmal cAMP receptor.

[Peptide components of Geolycosa spider venom modulate P2X receptor activity of rat sensory neurons].

Almost each natural venom comprises a considerable combinatorial library of bioactive substances that have been optimized during evolution. Particular attention is devoted currently on a search for new modulators of ion channels from the venoms of arthropods. We have studied the effect of peptidous compounds of the Lycosa spider venom on the activity of P2X receptors in DRG neurons of rats. As a result, at least 7 proteins modulating various P2X receptor-operated ionic currents in the sensory neurons of rats have been found.

[New cation channel of the T-lymphocyte nuclear membrane].

There are little data on the properties of ion channels in the inner nuclear membrane of lymphocytes, though the transport system between cytoplasm and karyoplasm is of a great importance for a complex genetic regulation in these cells. Using the patch-clamp technique we have investigated ion channels of the inner membrane of nuclei isolated from cultured T-lymphoblasts. Our research has shown that there are anionic (370 pS) and cationic (152 pS) channels on the inner nuclear membrane. The latter were characterized by fast kinetics and rapid fluctuations; they had high open probability and were inactivated at large negative potentials. These channels were permeable for K+ and Na+, but impermeable for Cl-. The physiological role of the channels is not clear, but they may play an important role in the ion balance between the cytoplasm and the lumen of the endoplasmatic reticulume of the cell.

Latrophilin, neurexin, and their signaling-deficient mutants facilitate alpha -latrotoxin insertion into membranes but are not involved in pore formation.

Pure alpha-latrotoxin is very inefficient at forming channels/pores in artificial lipid bilayers or in the plasma membrane of non-secretory cells. However, the toxin induces pores efficiently in COS-7 cells transfected with the heptahelical receptor latrophilin or the monotopic receptor neurexin. Signaling-deficient (truncated) mutants of latrophilin and latrophilin-neurexin hybrids also facilitate pore induction, which correlates with toxin binding irrespective of receptor structure. This rules out the involvement of signaling in pore formation. With any receptor, the alpha-latrotoxin pores are permeable to Ca(2+) and small molecules including fluorescein isothiocyanate and norepinephrine. Bound alpha-latrotoxin remains on the cell surface without penetrating completely into the cytosol. Higher temperatures facilitate insertion of the toxin into the plasma membrane, where it co-localizes with latrophilin (under all conditions) and with neurexin (in the presence of Ca(2+)). Interestingly, on subsequent removal of Ca(2+), alpha-latrotoxin dissociates from neurexin but remains in the membrane and continues to form pores. These receptor-independent pores are inhibited by anti-alpha-latrotoxin antibodies. Our results indicate that (i) alpha-latrotoxin is a pore-forming toxin, (ii) receptors that bind alpha-latrotoxin facilitate its insertion into the membrane, (iii) the receptors are not physically involved in the pore structure, (iv) alpha-latrotoxin pores may be independent of the receptors, and (v) pore formation does not require alpha-latrotoxin interaction with other neuronal proteins.

[Binding sites for A2 and A24 monoclonal antibodies on the alpha-latrotoxin molecule]. / Uchastki uznavaniia monoklonal'nykh antitel A4 i A24 v molekule alpha-latrotoksina.

alpha-Latrotoxin (alpha-LTX) binding sites to functionally active monoclonal antibodies (MA) A4 and A24 were localized using three approaches: hydrolysis of the toxin followed by the N-terminal sequencing of immunoreactive peptides; the study of antibody interaction with several recombinant alpha-LTX fragments; and Western immunoblotting of synthetic overlapping peptides (6-8 aa) whose structures correspond to that of the immunoreactive alpha-LTX fragment. It was shown that the MA A4 epitope is located within the F234-M294 protein fragment and that of MA A24 interacts with the fragment 347FDKDIT352.

[Molecular cloning and primary structure of cDNA fragment for alpha-latrocrustatoxin from black widow spider venom]. / Molekuliarnoe klonirovanie i opredelenie pervichnoi struktury fragmenta kDNK alpha-latrokrustotoksina iz iada pauka karakurta.

A fragment of the structural gene of alpha-latrocrustotoxin, a new representative of latrotoxins from black widow spider venom, was cloned. The fragment (1191 bp) was obtained by means of PCR based on the data obtained by sequencing tryptic peptides of the toxin. The fragment codes for a 397-aa sequence. The encoded polypeptide is the C-terminal fragment of the toxin central domain that presumably contains a site responsible for the toxin species specificity. The structural similarity of this fragment to the corresponding fragments of other latrotoxins was studied.

[Isolation and partial structural characteristics of major toxic components of Latrodectus pallidus venom]. / Vydelenie i chastichnaia strukturnaia kharakteristika osnovnykh toksicheskikh komponentov iada pauka Latrodectus pallidus.

Toxic components of the Latrodectus pallidus spider venom were isolated and characterized. The venom was shown to contain a toxin specific for mammals and at least one insectospecific toxin. Partial amino acid sequences of both toxins were determined, and their high structural homology with previously studied alpha-latrotoxin and alpha-latroinsectotoxin from L. mactans tredecimguttatus was found.

Functional expression of a recombinant unitary glutamate receptor from Xenopus, which contains N-methyl-D-aspartate (NMDA) and non-NMDA receptor subunits.

A cDNA encoding a 100-kDa subunit (XenNR1) of the N-methyl-D-aspartate (NMDA) glutamate receptor type has been cloned from Xenopus central nervous system. When XenNR1 is coexpressed in a mammalian cell line with a recently cloned 51-kDa non-NMDA receptor subunit (XenU1), also from Xenopus, it forms a functional unitary receptor exhibiting the pharmacological properties characteristic of both NMDA and non-NMDA receptors. Firstly, XenU1 can replace NR2 subunits, in complementing XenNR1 to introduce the ligand binding properties of a complete NMDA receptor. Second, responses to both NMDA and non-NMDA receptor agonists and antagonists were obtained in patch-clamp recordings from the cotransfected cells, but no significant responses were recorded when the cells were singly transfected. Third, from solubilized cell membranes from the cotransfected cells, an antibody to the NR1 subunit coprecipitated the binding sites of the non-NMDA receptor subunit. The unitary glutamate receptor has a unique set of properties that denote intersubunit interaction, including a glycine requirement for the responses to non-NMDA as well as to NMDA receptor agonists and voltage-dependent block by Mg2+ of the non-NMDA agonist responses.

Cloning and structure of delta-latroinsectotoxin, a novel insect-specific member of the latrotoxin family: functional expression requires C-terminal truncation.

The venom of the black widow spider (BWSV) (Latrodectus mactans tredecimguttatus) contains several potent, high molecular mass (>110 kDa) neurotoxins that cause neurotransmitter release in a phylum-specific manner. The molecular mechanism of action of these proteins is poorly understood because their structures are largely unknown, and they have not been functionally expressed. This study reports on the primary structure of delta-latroinsectotoxin (delta-LIT), a novel insect-specific toxin from BWSV, that contains 1214 amino acids. delta-LIT comprises four structural domains: a signal peptide followed by an N-terminal domain that exhibits the highest degree of identity with other latrotoxins, a central region composed of 15 ankyrin-like repeats, and a C-terminal domain. The domain organization of delta-LIT is similar to that of other latrotoxins, suggesting that these toxins are a family of related proteins. The predicted molecular mass and apparent mobility of the protein (approximately 130 kDa) encoded in the delta-LIT gene differs from that of native delta-LIT purified from BWSV (approximately 100 kDa), suggesting that the toxin is produced by proteolytic processing of a precursor. MALDI-MS of purified native delta-LIT revealed a molecular ion with m/z+ of 110916 +/- 100, indicating that the native delta-LIT is 991 amino acids in length. When the full-length delta-LIT cDNA was expressed in bacteria the protein product was inactive, but expression of a C-terminally truncated protein containing 991 residues produced a protein that caused massive neurotransmitter release at the locust neuromuscular junction at nanomolar concentrations. Channels formed in locust muscle membrane and artificial lipid bilayers by the native delta-LIT have a high Ca2+ permeability, whereas those formed by truncated, recombinant protein do not.

[Primary structure of delta-latroinsectotoxin from venom of the Latrodectus mactans tredecimguttatus spider]. / Pervichnaia struktura delta-latroinsektotoksina iz iada pauka Latrodectus mactans tredecimguttatus.

The structural gene of delta-latroinsectotoxin was cloned and its nucleotide sequence was determined. The gene contains an open reading frame of 3642 bp. The deduced amino acid sequence is homologous to the sequences of latrotoxins studied earlier.

Low molecular weight components from black widow spider venom.

Highly purified alpha-latrotoxin from the black widow spider venom (alpha-LTX) consists of two polypeptides with mol. wts of 130,000 and 8000 (LMWP). We have isolated two low mol. wt proteins LMWP and LMWP2 from the low mol. wt fraction of this venom. The chemical properties of these proteins and partial amino acid sequence of novel protein LMWP2 were studied. By means of i.v. or intracerebroventricular injections into mice it was shown that low mol. wt components of the venom at concentrations of 2.3 mg/kg and 0.8 mg/kg, respectively, did not possess any direct toxic effect on vertebrates. Injections of each protein into the third thoracic segment of cockroaches Periplaneta americana (doses up to 80 micrograms/g) did not cause lethality or paralysis of insects.

Modulation of functional activities of the neurotoxin from black widow spider venom.

We have studied the action of an alpha-latrotoxin (alpha-LTX) complex of two polypeptides (LTX 130 kDa and low molecular weight protein (LMWP) 8 kDa) and the action of a venom fraction containing LTX with excess LMWP on calcium influx into synaptosomes and PC12 cells as well as on [14C]GABA release from synaptosomes. Both preparations considerably activate calcium influx and stimulate [14C]GABA release from synaptosomes. Preincubation of both preparations with antibodies against a 14 amino acid residue C-terminal peptide of LMWP differentially modulates these effects. Antibodies inhibit induced calcium influx and enhance induced GABA release.

Polyamine spider toxins are potent un-competitive antagonists of rat cortex excitatory amino acid receptors.

We examined the effect of argiopine and argiopinine 3, low molecular weight polyamine venom components of the spider Argiope lobata, on rat cortical excitatory amino acid (EAA) receptors expressed in Xenopus oocytes. Responses to 100 microM N-methyl-D-aspartate (NMDA) with 10 microM glycine were blocked by both of the polyamine toxins in a dose-dependent manner. Both compounds had similar potencies against 100 microM kainate or 50 microM (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (L-AMPA). Oscillatory responses to 2 microM quisqualate were unaffected by either polyamine toxin. Increasing concentrations of either NMDA, kainate or AMPA were unable to overcome the antagonism by either spider toxin. We were able to demonstrate a use-dependent phenomenon similar to that of phencyclidine; neither polyamine toxin affected the NMDA, kainate or AMPA response without the presence of the respective agonist.

[Study of the amino acid sequence of latroinsectotoxin from black widow spider venom]. / Issledovanie aminokislotnoi posledovatel'nosti latroinsektotoksina iz iada pauka karakurta.

The N-terminal amino acid sequence of alpha-latroinsectotoxin from the venom of Latrodectus mactans tredecimguttatus was determined. Then the toxin was digested by trypsin and total or partial amino acid sequences of twenty-six tryptic peptides were established. This resulted in the structural information needed for the construction of probes followed by the cloning of the alpha-latroinsectotoxin structural gene.

[Structure of tryptic fragments of a neurotoxin from black widow spider venom]. / Struktura tripticheskikh fragmentov neirotoksina iz iada pauka karakurta.

The N-terminal amino acid sequence of a neurotoxin from the venom of Latrodectus mactans tredecimguttatus (alpha-latrotoxin) was determined. Latrotoxin was subjected to the tryptic cleavage and total or partial amino acid sequences of 25 peptides were established. In total the tryptic fragments contained 252 amino acid residues. Essential structural information on cloning of the latrotoxin structural gene was obtained.

[The development of protracted pneumonias in patients with a history of influenza and acute respiratory diseases]. / Razvitie zatiazhnykh pnevmonii u bol'nykh, perenesshikh gripp i ostrye respiratornye zabolevaniia.

A study is presented of 582 patients with acute viral-bacterial pneumonia in those with a history of influenza and acute respiratory disease (ARD). Protracted course of the disease was observed in 121 (20.8%) and 461 (79.2%) the course of pneumonia was acute. It is shown that the formation of protracted of acute pneumonia in patients with influenza and ARD is furthered by several factors: age, foci of chronic infection, a history of inflammation, increased level of circulating immune complexes, late hospitalization and inadequate therapy. Experiments on Syrian hamsters with induced parainfluenzal infection showed that mixed viral-bacterial infection is more severe than monoinfection.

[Argiopinin-binding proteins from the membrane of the bull cerebral cortex]. / Argiopininsviazyvaiushchii belok iz membran kory mozga byka.

The 40 kDa argiopinin-binding glycoprotein has been isolated from the solubilised preparations of bovine cerebrum membranes by means of two-step biospecific chromatography on affinity sorbents with immobilized glutamate and argiopinins. This receptor component displays a specific L-[3H]glutamate binding with Kd = 0.18 +/- +/- 0.019 mumole and Bmax = 43 +/- 4.5 nmole/mg. Amino acid analysis reveals it to be a member of integral membrane proteins.