Biomedical subjects

A N Akopian

Publications and source records attributed to A N Akopian.

At least 19 recordsLinked to original sources

PubMed2002

[Pathological types of breathing during acute hypoxia].

Different breathing pathologies (apneusis, gasping, Cheyne-Stokes breathing and Kussmaul breathing) were observed in anaesthetized rabbits and rats exposed to hypoxia at the altitude of 7500-8000 meters. Dominance of the high-voltage slow EKG activity (the delta-wave type) suggested deterioration of the brain functioning. Registration of impulse activities of the inspiration and expiration breathing neurons in medulla oblongata revealed a higher tolerance of the inspiration neurons to severe hypoxia which is attributed to the metabolic specifics and functional heterogeneity of these neurons. Character of the neuron impulsation is indicative of the reflectory effects of hemo- and mechanoreceptors, and the direct effects of different humoral substances resulting in impairment of the central control of pacemakers and breathing pathologies. It appears that various breathing pathologies in altitude chambers and mountains are triggered by the hypoxic factor. However, no dependence was established either between the initial breathing rhythm (before "ascent") and various types of breathing at the "altitude" or these types of breathing and magnitude of hypoxia.

Acute Disease↗

PubMed2000

Warm-coding deficits and aberrant inflammatory pain in mice lacking P2X3 receptors.

ATP activates damage-sensing neurons (nociceptors) and can evoke a sensation of pain. The ATP receptor P2X3 is selectively expressed by nociceptors and is one of seven ATP-gated, cation-selective ion channels. Here we demonstrate that ablation of the P2X3 gene results in the loss of rapidly desensitizing ATP-gated cation currents in dorsal root ganglion neurons, and that the responses of nodose ganglion neurons to ATP show altered kinetics and pharmacology resulting from the loss of expression of P2X(2/3) heteromultimers. Null mutants have normal sensorimotor function. Behavioural responses to noxious mechanical and thermal stimuli are also normal, although formalin-induced pain behaviour is reduced. In contrast, deletion of the P2X3 receptor causes enhanced thermal hyperalgesia in chronic inflammation. Notably, although dorsal-horn neuronal responses to mechanical and noxious heat application are normal, P2X3-null mice are unable to code the intensity of non-noxious 'warming' stimuli.

Adenosine Triphosphate↗

PubMed2000

A new member of the acid-sensing ion channel family.

Acid-sensing ion channels (ASICs) are members of the epithelial sodium channel (ENaC)-degenerin family of two-pass transmembrane segment protein subunits which form multimeric cation channels. Members of the ENaC-degenerin family are gated by stimuli as diverse as protons, peptides and mechanical distension. Here we describe a new member of the family, SPASIC or ASIC 4 (spinal cord ASIC) which is expressed throughout the central nervous system in an overlapping population of neurons that also express the ASIC subunit MDEG2. ASIC-4 which shows 44% identify with ASIC is developmentally regulated and expressed in a subset of sensory neurons as well as in the CNS. However, despite the strong homology with ASIC, the ASIC-4 transcript does not encode a proton gated cation channel.

Acid Sensing Ion Channels↗

PubMed1999

A novel persistent tetrodotoxin-resistant sodium current in SNS-null and wild-type small primary sensory neurons.

TTX-resistant (TTX-R) sodium currents are preferentially expressed in small C-type dorsal root ganglion (DRG) neurons, which include nociceptive neurons. Two mRNAs that are predicted to encode TTX-R sodium channels, SNS and NaN, are preferentially expressed in C-type DRG cells. To determine whether there are multiple TTX-R currents in these cells, we used patch-clamp recordings to study sodium currents in SNS-null mice and found a novel persistent voltage-dependent sodium current in small DRG neurons of both SNS-null and wild-type mice. Like SNS currents, this current is highly resistant to TTX (Ki = 39+/-9 microM). In contrast to SNS currents, the threshold for activation of this current is near 70 mV, the midpoint of steady-state inactivation is -44 +/- 1 mV, and the time constant for inactivation is 43+/-4 msec at 20 mV. The presence of this current in SNS-null and wild-type mice demonstrates that a distinct sodium channel isoform, which we suggest to be NaN, underlies this persistent TTX-R current. Importantly, the hyperpolarized voltage-dependence of this current, the substantial overlap of its activation and steady-state inactivation curves and its persistent nature suggest that this current is active near resting potential, where it may play an important role in regulating excitability of primary sensory neurons.

Animals↗

PubMed1999

Trans-splicing of a voltage-gated sodium channel is regulated by nerve growth factor.

Mammalian sensory neurons express a voltage-gated sodium channel named SNS. Here we report the identification of an SNS transcript (SNS-A) that contains an exact repeat of exons 12, 13 and 14 encoding a partial repeat of domain II. Because the exons 12-14 are present in single copies in genomic DNA, the SNS-A transcript must arise by trans-splicing. Nerve growth factor, which regulates pain thresholds, and the functional expression of voltage-gated sodium channels increases the levels of the SNS-A transcript several-fold both in vivo and in vitro as measured by RNase protection methods, as well as RT-PCR. These data demonstrate a novel regulatory role for the nerve growth factor and are the first example of trans-splicing in the vertebrate nervous system.

Alternative Splicing↗

PubMed1999

The tetrodotoxin-resistant sodium channel SNS has a specialized function in pain pathways.

Many damage-sensing neurons express tetrodotoxin (TTX)-resistant voltage-gated sodium channels. Here we examined the role of the sensory-neuron-specific (SNS) TTX-resistant sodium channel alpha subunit in nociception and pain by constructing sns-null mutant mice. These mice expressed only TTX-sensitive sodium currents on step depolarizations from normal resting potentials, showing that all slow TTX-resistant currents are encoded by the sns gene. Null mutants were viable, fertile and apparently normal, although lowered thresholds of electrical activation of C-fibers and increased current densities of TTX-sensitive channels demonstrated compensatory upregulation of TTX-sensitive currents in sensory neurons. Behavioral studies demonstrated a pronounced analgesia to noxious mechanical stimuli, small deficits in noxious thermoreception and delayed development of inflammatory hyperalgesia. These data show that SNS is involved in pain pathways and suggest that blockade of SNS expression or function may produce analgesia without side effects.

Afferent Pathways↗

PubMed1998

A sensory neuron-specific, proton-gated ion channel.

Proton-gated channels expressed by sensory neurons are of particular interest because low pH causes pain. Two proton-gated channels, acid-sensing ionic channel (ASIC) and dorsal root ASIC (DRASIC), that are members of the amiloride-sensitive ENaC/Degenerin family are known to be expressed by sensory neurons. Here, we describe the cloning and characterization of an ASIC splice variant, ASIC-beta, which contains a unique N-terminal 172 aa, as well as unique 5' and 3' untranslated sequences. ASIC-beta, unlike ASIC and DRASIC, is found only in a subset of small and large diameter sensory neurons and is absent from sympathetic neurons or the central nervous system. The patterns of expression of ASIC and ASIC-beta transcripts in rat dorsal root ganglion neurons are distinct. When expressed in COS-7 cells, ASIC-beta forms a functional channel with electrophysiological properties distinct from ASIC and DRASIC. The pH dependency and sensitivity to amiloride of ASIC-beta is similar to that described for ASIC, but unlike ASIC, the channel is not permeable to calcium, nor are ASIC-beta-mediated currents inhibited by extracellular calcium. The unique distribution of ASIC-beta suggests that it may play a specialized role in sensory neuron function.

Acid Sensing Ion Channels↗

PubMed1997

Structure and chromosomal mapping of the mouse P2X3 gene.

P2X3 is one of seven cloned ATP-gated non-selective cation channels. We have isolated a full-length mouse P2X3 gene from a phage lambda-129/Sv genomic library. The gene consists of 12 exons spanning a locus of approximately 40 kb. No significant similarities have been found between the genomic organisation of the mouse P2X3 gene and genes encoding other ion channels. The encoded mouse P2X3 protein consists of 397 amino acids and shows 99% identity with rat P2X3. Using RNase protection and primer extension assays, multiple transcription initiation sites have been mapped in the mouse P2X3 promoter to a region 162-168 bp upstream of the translation initiation codon. The P2X3 gene has been mapped to mouse chromosome 2p by fluorescence in situ hybridisation. The RAG locus-associated gene T160 is located 1.8 kb upstream of the transcription start site of mouse P2X3 gene. The promoter region of the mouse P2X3 gene lacks a conventional TATA and CCAAT consensus sites, and initiator elements. P2X3 is the first member of the P2X gene family to be completely characterised.

Amino Acid Sequence↗

PubMed1997

A single serine residue confers tetrodotoxin insensitivity on the rat sensory-neuron-specific sodium channel SNS.

Sensory neurons express a sodium channel (SNS) that is highly resistant to block by tetrodotoxin (IC50 = 60 microM). SNS is 65% homologous to the cardiac sodium channel, in which a single hydrophilic residue in the SS2 segment is critical for tetrodotoxin resistance. By site-directed mutagenesis, we have substituted phenylalanine for serine at the equivalent position in SNS: this mutated (S356F) SNS channel is functionally similar to wild-type SNS when expressed in Xenopus oocytes, but is potently blocked by tetrodotoxin and saxitoxin with IC50s of 2.8 nM and 8.2 nM, respectively. These data provide clues to the rational design of selective blockers of SNS with potential as analgesic drugs.

Animals↗

PubMed1997

Cloning and characterization of a mouse sensory neuron tetrodotoxin-resistant voltage-gated sodium channel gene, Scn10a.

Small-diameter sensory neurons associated with unmyelinated axons express a tetrodotoxin-insensitive (TTXi) voltage-gated sodium channel (VGSC) that may play an important role in the transmission of nociceptive information to the spinal cord. A TTXi VGSC, named SNS, that accounts for the tetrodotoxin-resistant sodium current described in sensory neurons has been cloned from rat dorsal root ganglia. Using recombinant lambda phage clones encoding a mouse 129/SV genomic library, we have determined the detailed structure of the mouse SNS gene (Scn10a), including the location of exon-intron boundaries and the nucleotide sequence of the exons. The gene consists of 27 exons spanning approximately 90 kb on chromosome 9. Mouse SNS shows 95.3% overall amino acid identity to rat SNS and 98.5% identity throughout the putative transmembrane segments and the intracellular loop linking domains 3 and 4. The sizes of the exons and the exon-intron junction positions of the mouse SNS and the human skeletal muscle VGSC genes are remarkably conserved. These results provide the basis for an evolutionary comparison of sodium channels, the construction and analysis of a mouse SNS null mutant as a direct approach to understanding the biological function of SNS, and the identification of regulatory elements that are responsible for the tissue- and cell-specific expression of SNS.

Animals↗

PubMed1997

A role for calcineurin in the desensitization of the P2X3 receptor.

The sensory neurone-specific ATP-gated cation channel P2X3, when expressed in Xenopus oocytes, desensitizes rapidly. Complete removal of extracellular calcium abolishes desensitization. Pretreatment of oocytes with cyclosporin also abolishes P2X3 desensitization. When the calcineurin auto-inhibitory peptide (CaN A457-481) was injected into oocytes, the rate of desensitization of P2X3 decreased on consecutive applications of ATP, suggesting a role for calcineurin in the regulation of desensitization. Truncated P2X3 clones initiating at Met-75 lack the N-terminal intracellular region, but express functional channels in oocytes that do not desensitize. Taken together, these data suggest that P2X3 desensitizes through a calcium-dependent calcineurin-mediated dephosphorylation involving N-terminal residues that are phosphorylated in functional channels.

Animals↗

PubMed1997

Structure and distribution of a broadly expressed atypical sodium channel.

A cDNA clone isolated from a rat dorsal root ganglion library encodes a 195 kDa voltage-gated sodium channel-like protein (SCL-11) with homology to the mouse (87%) and human (72%) atypical Na+ channels and rat partial clone NaG (98%). Two dominant mRNAs of 4.5 and 7 kb are expressed. The transcripts are present in lung, Schwann cells, pituitary and tissues containing smooth muscle cells. No functional channels could be detected on oocyte injection with cRNA, consistent with the absence of structural features necessary for voltage-gated sodium channel activity.

Amino Acid Sequence↗

PubMed1997

Regulation of expression of the sensory neuron-specific sodium channel SNS in inflammatory and neuropathic pain.

Increased voltage-gated sodium channel activity may contribute to the hyperexcitability of sensory neurons in inflammatory and neuropathic pain states. We examined the levels of the transcript encoding the tetrodotoxin-resistant sodium channel SNS in dorsal root ganglion neurons in a range of inflammatory and neuropathic pain models in the rat. Local Freund's adjuvant or systemic nerve growth factor-induced inflammation did not substantially alter the total levels of SNS mRNA. When NGF-treated adult rat DRG neurons in vitro were compared with NGF-depleted control neurons, SNS total mRNA levels and the levels of membrane-associated immunoreactive SNS showed a small increase (17 and 25%, respectively), while CGRP levels increased fourfold. SNS expression is thus little dependent on NGF even though SNS transcript levels dropped by more than 60% 7-14 days after axotomy. In the streptozotocin diabetic rat SNS levels fell 25%, while in several manipulations of the L5/6 tight nerve ligation rat neuropathic pain model, SNS levels fell 40-80% in rat strains that are either susceptible or relatively resistant to the development of allodynia. Increased expression of SNS mRNA is thus unlikely to underlie sensory neuron hyperexcitability associated with inflammation, while lowered SNS transcript levels are associated with peripheral nerve damage.

Animals↗

PubMed1996

A tetrodotoxin-resistant voltage-gated sodium channel expressed by sensory neurons.

Dorsal root ganglion sensory neurons associated with C-fibres, many of which are activated by tissue-damage, express an unusual voltage-gated sodium channel that is resistant to tetrodotoxin. We report here that we have identified a 1,957 amino-acid sodium channel in these cells that shows 65% identity with the rat cardiac tetrodotoxin-insensitive sodium channel, and is not expressed in other peripheral and central neurons, glia or non-neuronal tissues. In situ hybridization shows that the channel is expressed only by small-diameter sensory neurons in neonatal and adult dorsal root and trigeminal ganglia. The channel is resistant to tetrodotoxin when expressed in Xenopus oocytes. The electrophysiological and pharmacological properties of the expressed channel are similar to those described for the small-diameter sensory neuron tetrodotoxin-resistant sodium channels. As some noxious input into the spinal cord is resistant to tetrodotoxin, block of expression or function of such a C-fibre-restricted sodium channel may have a selective analgesic effect.

Amino Acid Sequence↗

PubMed1996

Molecular genetic approaches to nociceptor development and function.

The activation of peripheral nociceptors is the subject of intense scrutiny, because of its significance in pain regulation. Genetic approaches, including homology cloning, difference cloning and transgenic manipulation of mice are providing useful insights into nociceptor function. Recent work suggests that transcriptional regulators (for example, islet-I), which are expressed relatively selectively in sensory neurones, play a crucial role in defining cellular phenotype. Difference cloning has identified genes which encode both ligand-gated and voltage-gated ion channels expressed by small-diameter sensory neurones. The role of inflammatory mediators such as NGF in regulating nociceptor function has been clarified in mis-expression and deletion studies. An understanding of the mechanisms that regulate gene expression in nociceptors should provide new ways to manipulate nociceptor sensitivity, with potential significance for pain therapy.

Animals↗

PubMed1996

[Cloning genes, specifically expressed in the cerebral cortex and cerebellum of adult rats].

To isolate genes differentially expressed in the rat brain cortex and cerebellum, a subtractive cDNA cloning procedure requiring only small quantities of poly(A+) RNA, followed by differential screening, was used. Two novel genes, MK and 3L7, with cortex- and cerebellum-specific expression were identified. These genes displayed different expression patterns in the brain cortex, cerebellum, and hippocampus during postnatal development.

Animals↗

PubMed1996

[Cloning of genes, differentially expressed in the cerebellum during postnatal rat development].

To identify genes differentially expressed in the developing rat cerebellum, a subtractive cDNA cloning procedure, followed by differential screening, was used. Four novel genes, MB, MF, 3E7, and 3C6, the transcriptional activity of which changed by a factor of five during cerebellar development, were isolated. The genes obtained were differentially expressed in different regions of the central nervous system. Variations in the levels of corresponding transcripts in the brain cortex and cerebellum were also recorded during postnatal rat development.

Animals↗

PubMed1995

A P2X purinoceptor expressed by a subset of sensory neurons.

ATP is known to depolarize sensory neurons, and may play a role in nociceptor activation when released from damaged tissue. Here we report the molecular cloning and characterization of a new member of the P2X receptor family, P2X3, expressed by these cells. The channel transcript was present in a subset of rat dorsal-root-ganglion sensory neurons, some of which express nociceptor-associated markers; it was absent in other tissues that were tested, including sympathetic, enteric and central nervous system neurons. Moreover, when expressed in Xenopus oocytes, the channel showed an ATP-dependent cation flux. P2X3 is the only ligand-gated channel known to be expressed exclusively by a subset of sensory neurons. The remarkable selectivity of expression of the channel coupled with its sensory neuron-like pharmacology suggests that this channel may transduce ATP-evoked nociceptor activation.

Amino Acid Sequence↗