Stephen H. Hadley

The latent human herpesvirus-6A genome specifically integrates in telomeres of human chromosomes in vivo and in vitro

Previous research has suggested that human herpesvirus-6 (HHV-6) may integrate into host cell chromosomes and be vertically transmitted in the germ line, but the evidence—primarily fluorescence in situ hybridization (FISH)—is indirect. We sought, first, to definitively test these two hypotheses. Peripheral blood mononuclear cells (PBMCs) were isolated from families in which several members, including at least one parent and child, had unusually high copy numbers of HHV-6 DNA per milliliter of blood. FISH confirmed that HHV-6 DNA colocalized with telomeric regions of one allele on chromosomes 17p13.3, 18q23, and 22q13.3, and that the integration site was identical among members of the same family. Integration of the HHV-6 genome into TTAGGG telomere repeats was confirmed by additional methods and sequencing of the integration site. Partial sequencing of the viral genome identified the same integrated HHV-6A strain within members of families, confirming vertical transmission of the viral genome. We next asked whether HHV-6A infection of naïve cell lines could lead to integration. Following infection of naïve Jjhan and HEK-293 cell lines by HHV-6, the virus integrated into telomeres. Reactivation of integrated HHV-6A virus from individuals’ PBMCs as well as cell lines was successfully accomplished by compounds known to induce latent herpesvirus replication. Finally, no circular episomal forms were detected even by PCR. Taken together, the data suggest that HHV-6 is unique among human herpesviruses: it specifically and efficiently integrates into telomeres of chromosomes during latency rather than forming episomes, and the integrated viral genome is capable of producing virions.

Ketamine, But Not Phencyclidine, Selectively Modulates Cerebellar GABAA Receptors Containing α6 and δ Subunits

Phencyclidine (PCP) and ketamine are dissociative anesthetics capable of inducing analgesia, psychomimetic behavior, and a catatonic state of unconsciousness. Despite broad similarities, there are notable differences between the clinical actions of ketamine and PCP. Ketamine has a lower incidence of adverse effects and generally produces greater CNS depression than PCP. Both noncompetitively inhibit NMDA receptors, yet there is little evidence that these drugs affect GABAA receptors, the primary target of most anesthetics. α6β2/3δ receptors are subtypes of the GABAA receptor family and are abundantly expressed in granular neurons within the adult cerebellum. Here, using an oocyte expression system, we show that at anesthetically relevant concentrations, ketamine, but not PCP, modulates α6β2δ and α6β3δ receptors. Additionally, at higher concentrations, ketamine directly activates these GABAA receptors. Comparatively, dizocilpine (MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine maleate]), a potent noncompetitive antagonist of NMDA receptors that is structurally unrelated to PCP, did not produce any effect on α6β2δ receptors. Of the recombinant GABAA receptor subtypes examined (α1β2, α1β2γ2, α1β2δ, α4β2γ2, α4β2δ, α6β2γ2, α6β2δ, and α6β3δ), the actions of ketamine were unique to α6β2δ and α6β3δ receptors. In dissociated granule neurons and cerebellar slice recordings, ketamine potentiated the GABAergic conductance arising from α6-containing GABAA receptors, whereas PCP showed no effect. Furthermore, ketamine potentiation was absent in cerebellar granule neurons from transgenic functionally null α6−/− and δ−/−mice. These findings suggest that the higher CNS depressant level achieved by ketamine may be the result of its selective actions on α6β2/3δ receptors.

Rat α6β2δ GABAA receptors exhibit two distinct and separable agonist affinities

The onset of motor learning in rats coincides with exclusive expression of GABAA receptors containing α6 and δ subunits in the granule neurons of the cerebellum. This development temporally correlates with the presence of a spontaneously active chloride current through α6‐containing GABAA receptors, known as tonic inhibition. Here we report that the coexpression of α6, β2, and δ subunits produced receptor–channels which possessed two distinct and separable states of agonist affinity, one exhibiting micromolar and the other nanomolar affinities for GABA. The high‐affinity state was associated with a significant level of spontaneous channel activity. Increasing the level of expression or the ratio of β2 to α6 and δ subunits increased the prevalence of the high‐affinity state. Comparative studies of α6β2δ, α1β2δ, α6β2γ2, α1β2γ2 and α4β2δ receptors under equivalent levels of expression demonstrated that the significant level of spontaneous channel activity is uniquely attributable to α6β2δ receptors. The pharmacology of spontaneous channel activity arising from α6β2δ receptor expression corresponded to that of tonic inhibition. For example, GABAA receptor antagonists, including furosemide, blocked the spontaneous current. Further, the neuroactive steroid 5α‐THDOC and classical glycine receptor agonists β‐alanine and taurine directly activated α6β2δ receptors with high potency. Specific mutation within the GABA‐dependent activation domain (βY157F) impaired both low‐ and high‐affinity components of GABA agonist activity in α6βY157Fδ receptors, but did not attenuate the spontaneous current. In comparison, a mutation located between the second and third transmembrane segments of the δ subunit (δR287M) significantly diminished the nanomolar component and the spontaneous activity. The possibility that the high affinity state of the α6β2δ receptor modulates the granule neuron activity as well as potential mechanisms affecting its expression are discussed.

Sensory Nerve Terminal Mitochondrial Dysfunction Activates Airway Sensory Nerves via Transient Receptor Potential (TRP) Channels

Mitochondrial dysfunction and subsequent oxidative stress has been reported for a variety of cell types in inflammatory diseases. Given the abundance of mitochondria at the peripheral terminals of sensory nerves and the sensitivity of transient receptor potential (TRP) ankyrin 1 (A1) and TRP vanilloid 1 (V1) to reactive oxygen species (ROS) and their downstream products of lipid peroxidation, we investigated the effect of nerve terminal mitochondrial dysfunction on airway sensory nerve excitability. Here we show that mitochondrial dysfunction evoked by acute treatment with antimycin A (mitochondrial complex III Qi site inhibitor) preferentially activated TRPA1-expressing “nociceptor-like” mouse bronchopulmonary C-fibers. Action potential discharge was reduced by the TRPA1 antagonist HC-030031. Inhibition of TRPV1 further reduced C-fiber activation. In mouse dissociated vagal neurons, antimycin A induced Ca2+ influx that was significantly reduced by pharmacological inhibition or genetic knockout of either TRPA1 or TRPV1. Inhibition of both TRPA1 and TRPV1 was required to abolish antimycin A-induced Ca2+ influx in vagal neurons. Using an HEK293 cell expression system, antimycin A induced concentration-dependent activation of both hTRPA1 and hTRPV1 but failed to activate nontransfected cells. Myxothiazol (complex III Qo site inhibitor) inhibited antimycin A-induced TRPA1 activation, as did the reducing agent dithiothreitol. Scavenging of both superoxide and hydrogen peroxide inhibited TRPA1 activation following mitochondrial modulation. In conclusion, we present evidence that acute mitochondrial dysfunction activates airway sensory nerves preferentially via TRPA1 through the actions of mitochondrially-derived ROS. This represents a novel mechanism by which inflammation may be transduced into nociceptive electrical signaling.

Rat alpha6beta2delta GABAA receptors exhibit two distinct and separable agonist affinities.

The onset of motor learning in rats coincides with exclusive expression of GABAA receptors containing α6 and δ subunits in the granule neurons of the cerebellum. This development temporally correlates with the presence of a spontaneously active chloride current through α6‐containing GABAA receptors, known as tonic inhibition. Here we report that the coexpression of α6, β2, and δ subunits produced receptor–channels which possessed two distinct and separable states of agonist affinity, one exhibiting micromolar and the other nanomolar affinities for GABA. The high‐affinity state was associated with a significant level of spontaneous channel activity. Increasing the level of expression or the ratio of β2 to α6 and δ subunits increased the prevalence of the high‐affinity state. Comparative studies of α6β2δ, α1β2δ, α6β2γ2, α1β2γ2 and α4β2δ receptors under equivalent levels of expression demonstrated that the significant level of spontaneous channel activity is uniquely attributable to α6β2δ receptors. The pharmacology of spontaneous channel activity arising from α6β2δ receptor expression corresponded to that of tonic inhibition. For example, GABAA receptor antagonists, including furosemide, blocked the spontaneous current. Further, the neuroactive steroid 5α‐THDOC and classical glycine receptor agonists β‐alanine and taurine directly activated α6β2δ receptors with high potency. Specific mutation within the GABA‐dependent activation domain (βY157F) impaired both low‐ and high‐affinity components of GABA agonist activity in α6βY157Fδ receptors, but did not attenuate the spontaneous current. In comparison, a mutation located between the second and third transmembrane segments of the δ subunit (δR287M) significantly diminished the nanomolar component and the spontaneous activity. The possibility that the high affinity state of the α6β2δ receptor modulates the granule neuron activity as well as potential mechanisms affecting its expression are discussed.

Sensory Nerve Terminal Mitochondrial Dysfunction Induces Hyperexcitability in Airway Nociceptors via Protein Kinase C

Airway sensory nerve excitability is a key determinant of respiratory disease-associated reflexes and sensations such as cough and dyspnea. Inflammatory signaling modulates mitochondrial function and produces reactive oxygen species (ROS). Peripheral terminals of sensory nerves are densely packed with mitochondria; thus, we hypothesized that mitochondrial modulation would alter neuronal excitability. We recorded action potential firing from the terminals of individual bronchopulmonary C-fibers using a mouse ex vivo lung-vagal ganglia preparation. C-fibers were characterized as nociceptors or non-nociceptors based upon conduction velocity and response to transient receptor potential (TRP) channel agonists. Antimycin A (mitochondrial complex III Qi site inhibitor) had no effect on the excitability of non-nociceptors. However, antimycin A increased excitability in nociceptive C-fibers, decreasing the mechanical threshold by 50% and increasing the action potential firing elicited by a P2X2/3 agonist to 270% of control. Antimycin A–induced nociceptor hyperexcitability was independent of TRP ankyrin 1 or TRP vanilloid 1 channels. Blocking mitochondrial ATP production with oligomycin or myxothiazol had no effect on excitability. Antimycin A–induced hyperexcitability was dependent on mitochondrial ROS and was blocked by intracellular antioxidants. ROS are known to activate protein kinase C (PKC). Antimycin A–induced hyperexcitability was inhibited by the PKC inhibitor bisindolylmaleimide (BIM) I, but not by its inactive analog BIM V. In dissociated vagal neurons, antimycin A caused ROS-dependent PKC translocation to the membrane. Finally, H2O2 also induced PKC-dependent nociceptive C-fiber hyperexcitability and PKC translocation. In conclusion, ROS evoked by mitochondrial dysfunction caused nociceptor hyperexcitability via the translocation and activation of PKC.

Characterization of cardiovascular reflexes evoked by airway stimulation with allylisothiocyanate, capsaicin, and ATP in Sprague-Dawley rats

Acute inhalation of airborne pollutants alters cardiovascular function and evidence suggests that pollutant-induced activation of airway sensory nerves via the gating of ion channels is critical to these systemic responses. Here, we have investigated the effect of capsaicin [transient receptor potential (TRP) vanilloid 1 (TRPV1) agonist], AITC [TRP ankyrin 1 (TRPA1) agonist], and ATP (P2X2/3 agonist) on bronchopulmonary sensory activity and cardiovascular responses of conscious Sprague-Dawley (SD) rats. Single fiber recordings show that allyl isothiocyanate (AITC) and capsaicin selectively activate C fibers, whereas subpopulations of both A and C fibers are activated by stimulation of P2X2/3 receptors. Inhalation of the agonists by conscious rats caused significant bradycardia, atrioventricular (AV) block, and prolonged PR intervals, although ATP-induced responses were lesser than those evoked by AITC or capsaicin. Responses to AITC were inhibited by the TRP channel blocker ruthenium red and the muscarinic antagonist atropine. AITC inhalation also caused a biphasic blood pressure response: a brief hypertensive phase followed by a hypotensive phase. Atropine accentuated the hypertensive phase, while preventing the hypotension. AITC-evoked bradycardia was not abolished by terazosin, the α1-adrenoceptor inhibitor, which prevented the hypertensive response. Anesthetics had profound effects on AITC-evoked bradycardia and AV block, which was abolished by urethane, ketamine, and isoflurane. Nevertheless, AITC inhalation caused bradycardia and AV block in paralyzed and ventilated rats following precollicular decerebration. In conclusion, we provide evidence that activation of ion channels expressed on nociceptive airway sensory nerves causes significant cardiovascular effects in conscious SD rats via reflex modulation of the autonomic nervous system.

Store-operated calcium entry in vagal sensory nerves is independent of Orai channels.

Vagal sensory nerves innervate the majority of visceral organs (e.g., heart, lungs, GI tract, etc) and their activation is critical for defensive and regulatory reflexes. Intracellular Ca(2+) is a key regulator of neuronal excitability and is largely controlled by the Ca(2+) stores of the endoplasmic reticulum. In other cell types store-operated channels (SOC) have been shown to contribute to the homeostatic control of intracellular Ca(2+). Here, using Ca(2+) imaging, we have shown that ER depletion in vagal sensory neurons (using thapsigargin or caffeine) in the absence of extracellular Ca(2+) evoked Ca(2+) influx upon re-introduction of Ca(2+) into the extracellular buffer. This store-operated Ca(2+) entry (SOCE) was observed in approximately 25-40% of vagal neurons, equally distributed among nociceptive and non-nociceptive sensory subtypes. SOCE was blocked by Gd(3+) but not by the Orai channel blocker SKF96365. We found Orai channel mRNA in extracts from whole vagal ganglia, but when using single cell RT-PCR analysis we found only 3 out of 34 neurons expressed Orai channel mRNA, indicating that Orai channel expression in the vagal ganglia was likely derived from non-neuronal cell types. Confocal microscopy of vagal neurons in 3 day cultures demonstrated rich ER tracker fluorescence throughout axonal and neurite structures and ER store depletion (thapsigargin) evoked Ca(2+) transients from these structures. However, no SOCE could be detected in the axonal/neurite structures of vagal neurons. We conclude that SOCE occurs in vagal sensory neuronal cell bodies through non-Orai mechanisms but is absent at nerve terminals.

Rat α6β2δ GABAAreceptors exhibit two distinct and separable agonist affinities: High-affinity and low-affinity states of α6β2δ receptors

The onset of motor learning in rats coincides with exclusive expression of GABAA receptors containing α6 and δ subunits in the granule neurons of the cerebellum. This development temporally correlates with the presence of a spontaneously active chloride current through α6‐containing GABAA receptors, known as tonic inhibition. Here we report that the coexpression of α6, β2, and δ subunits produced receptor–channels which possessed two distinct and separable states of agonist affinity, one exhibiting micromolar and the other nanomolar affinities for GABA. The high‐affinity state was associated with a significant level of spontaneous channel activity. Increasing the level of expression or the ratio of β2 to α6 and δ subunits increased the prevalence of the high‐affinity state. Comparative studies of α6β2δ, α1β2δ, α6β2γ2, α1β2γ2 and α4β2δ receptors under equivalent levels of expression demonstrated that the significant level of spontaneous channel activity is uniquely attributable to α6β2δ receptors. The pharmacology of spontaneous channel activity arising from α6β2δ receptor expression corresponded to that of tonic inhibition. For example, GABAA receptor antagonists, including furosemide, blocked the spontaneous current. Further, the neuroactive steroid 5α‐THDOC and classical glycine receptor agonists β‐alanine and taurine directly activated α6β2δ receptors with high potency. Specific mutation within the GABA‐dependent activation domain (βY157F) impaired both low‐ and high‐affinity components of GABA agonist activity in α6βY157Fδ receptors, but did not attenuate the spontaneous current. In comparison, a mutation located between the second and third transmembrane segments of the δ subunit (δR287M) significantly diminished the nanomolar component and the spontaneous activity. The possibility that the high affinity state of the α6β2δ receptor modulates the granule neuron activity as well as potential mechanisms affecting its expression are discussed.