Allen C. Myers

Interganglionic segregation of distinct vagal afferent fibre phenotypes in guinea-pig airways

1. The present study addressed the hypothesis that jugular and nodose vagal ganglia contain the somata of functionally and anatomically distinct airway afferent fibres. 2. Anatomical investigations were performed by injecting guinea‐pig airways with the neuronal tracer Fast Blue. The animals were killed 7 days later, and the ganglia were removed and immunostained with antisera against substance P (SP) and neurofilament protein (NF). In the nodose ganglion, NF‐immunoreactive neurones accounted for about 98% of the Fast Blue‐labelled cells while in the jugular ganglion they accounted for approximately 48%. SP and NF immunoreactivity was never (n = 100) observed in the same cell suggesting that the antisera labelled distinct populations. 3. Electrophysiological investigations were performed using an in vitro guinea‐pig tracheal and bronchial preparation with intact afferent vagal pathways, including nodose and jugular ganglia. Action potentials arriving from single airway afferent nerve endings were monitored extracellularly using a glass microelectrode positioned near neuronal cell bodies in either ganglion. 4. The nodose ganglion contained the somata of mainly fast‐conducting tracheal A delta fibres whereas the jugular ganglion contained equal numbers of C fibre and A delta fibre tracheal afferent somata. The nodose A delta neurones adapted rapidly to mechanical stimulation, had relatively low mechanical thresholds, were not activated by capsaicin and adapted rapidly to a hyperosmotic stimulus. By contrast, jugular A delta and C fibres adapted slowly to mechanical stimulation, were often activated by capsaicin, had higher mechanical thresholds and displayed a slow adaptation to a hyperosmotic stimulus. 5. The anatomical, physiological and pharmacological data provide evidence to support the contention that the vagal ganglionic source of the fibre supplying the airways ultimately dictates its neurochemical and physiological phenotype.

Nrf2-regulated glutathione recycling independent of biosynthesis is critical for cell survival during oxidative stress

Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) is the primary transcription factor protecting cells from oxidative stress by regulating cytoprotective genes, including the antioxidant glutathione (GSH) pathway. GSH maintains cellular redox status and affects redox signaling, cell proliferation, and death. GSH homeostasis is regulated by de novo synthesis as well as GSH redox state; previous studies have demonstrated that Nrf2 regulates GSH homeostasis by affecting de novo synthesis. We report that Nrf2 modulates the GSH redox state by regulating glutathione reductase (GSR). In response to oxidants, lungs and embryonic fibroblasts (MEFs) from Nrf2-deficient (Nrf2(-/-)) mice showed lower levels of GSR mRNA, protein, and enzyme activity relative to wild type (Nrf2(+/+)). Nrf2(-/-) MEFs exhibited greater accumulation of glutathione disulfide and cytotoxicity compared to Nrf2(+/+) MEFs in response to t-butylhydroquinone, which was rescued by restoring GSR. Microinjection of glutathione disulfide induced greater apoptosis in Nrf2(-/-) MEFs compared to Nrf2(+/+) MEFs. In silico promoter analysis of the GSR gene revealed three putative antioxidant-response elements (ARE1, -44; ARE2, -813; ARE3, -1041). Reporter analysis, site-directed mutagenesis, and chromatin immunoprecipitation assays demonstrated binding of Nrf2 to two AREs distal to the transcription start site. Overall, Nrf2 is critical for maintaining the GSH redox state via transcriptional regulation of GSR and protecting cells against oxidative stress.

Subtypes of vagal afferent C‐fibres in guinea‐pig lungs

An ex vivo, vagally innervated, lung preparation was used to address the hypothesis that vagal C‐fibres comprise at least two distinct phenotypes. Histological and extracellular electrophysiological experiments revealed that vagal C‐fibres innervating the pulmonary system are derived from cell bodies situated in two distinct vagal sensory ganglia. The jugular (superior) ganglion neurones project C‐fibres to both the extrapulmonary airways (larynx, trachea and bronchus) and the lung parenchymal tissue. By contrast, C‐fibres from nodose (inferior) neurones innervate primarily structures within the lungs. Histologically, nodose neurones projecting lung C‐fibres were different from the jugular neurones in that they were significantly less likely to express neurokinins. The nerve terminals within the lungs of both nodose and jugular C‐fibres responded with action potential discharge to capsaicin and bradykinin application, but only the nodose C‐fibre population responded with action potential discharge to the P2X selective receptor agonist α,β‐methylene‐ATP. Whole cell patch clamp recording of capsaicin‐sensitive nodose and jugular ganglion neurones retrogradely labelled from the lung tissue revealed that, like the nerve terminals, lung specific nodose C‐fibre neurones express functional P2X receptors, whereas lung specific jugular C‐fibres do not. The data support the hypothesis that both neural crest‐derived neurones (jugular ganglia) and placode‐derived neurones (nodose ganglia) project C‐fibres in the vagus, and that these two C‐fibre populations represent distinct phenotypes.

Immunomodulation of afferent neurons in guinea-pig isolated airway.

1. The trachea, larynx and main bronchi with the right vagus nerve and nodose ganglion were isolated from guinea‐pigs passively immunized 24 h previously with serum containing anti‐ovalbumin antibody. 2. The airways were placed in one compartment of a Perspex chamber for recording of isometric tension while the nodose ganglion and attached vagus nerve were pulled into another compartment. Action potentials arriving from single airway afferent nerve endings were monitored extracellularly using a glass microelectrode positioned near neuronal cell bodies in the ganglion. Mechanosensitivity of the nerve endings was quantified using calibrated von Frey filaments immediately before and after exposure to antigen (10 micrograms ml‐1 ovalbumin). 3. Ten endings responded to the force exerted by the lowest filament (0.078 mN) and were not further investigated. In airways from thirteen immunized guinea‐pigs, the mechanical sensitivity of A delta afferent fibres (conduction velocity = 4.3 +/‐ 0.6 m s‐1) was enhanced 4.1 +/‐ 0.9‐fold following airway exposure to antigen (P < 0.005). Mechanical sensitivities of afferent fibres (conduction velocity = 4.3 +/‐ 0.6 m s‐1) from non‐immunized control guinea‐pig airways were unaffected by antigen (n = 13). 4. Antigen did not overtly cause action potential generation except in one instance when the receptive field was located over the smooth muscle. This ending also responded to methacholine suggesting that spatial changes in the receptive field, induced by muscle contraction, were responsible for the activation. 5. The mediators responsible for these effects are unknown, although histamine, prostaglandins, leukotrienes and tachykinins do not appear to be essential. The increase in mechanical responsiveness was not associated with the smooth muscle contraction since leukotriene C4, histamine and tachykinins, which all caused a similar contraction to antigen, did not affect mechanical thresholds. Moreover, the antigen‐induced increases in excitability persisted beyond the duration of the smooth muscle contraction. 6. These results demonstrate that antigen‐antibody‐mediated inflammatory processes may enhance the excitability of vagal afferent nerve terminals projecting from the airway and thus may contribute to the pathophysiology of allergic airway diseases.

Heightened endoplasmic reticulum stress in the lungs of patients with chronic obstructive pulmonary disease: the role of Nrf2-regulated proteasomal activity

RATIONALE Nuclear factor erythroid 2-related factor 2 (Nrf2), an important regulator of lung antioxidant defenses, declines in chronic obstructive pulmonary disease (COPD). However, Nrf2 also regulates the proteasome system that degrades damaged and misfolded proteins. Because accumulation of misfolded proteins in the endoplasmic reticulum (ER) causes ER stress and ER stress-induced apoptosis, Nrf2 may potentially prevent ER stress-mediated apoptosis in COPD. OBJECTIVES To determine whether Nrf2-regulated proteasome function affects ER stress-mediated apoptosis in COPD. METHODS We assessed the expression of Nrf2, Nrf2-dependent proteasomal subunits, proteasomal activity, markers of ER stress, and apoptosis in emphysematous lungs of mice exposed to cigarette smoke (CS) as well as peripheral lung tissues from normal control subjects and patients with COPD. MEASUREMENTS AND MAIN RESULTS Compared with wild-type mice, emphysematous lungs of CS-exposed Nrf2-deficient mice exhibited markedly lower proteasomal activity and elevated markers of ER stress and apoptosis. Furthermore, compared with normal control subjects, lungs of patients with mild and advanced COPD showed a marked decrease in the expression of Nrf2-regulated proteasomal subunits and total proteasomal activity. However, they were associated with greater levels of ER stress and apoptosis markers. In vitro studies have demonstrated that enhancing proteasomal activity in Beas2B cells either by sulforaphane, an activator of Nrf2, or overexpression of Nrf2-regulated proteasomal subunit PSMB6, significantly inhibited cigarette smoke condensate (CSC)-induced ER stress and cell death. CONCLUSIONS Impaired Nrf2 signaling causes significant decline in proteasomal activity and heightens ER stress response in lungs of patients with COPD and CS-exposed mice. Accordingly, pharmacological approaches that augment Nrf2 activity may protect against COPD progression by both up-regulating antioxidant defenses and relieving ER stress.

Electrophysiological effects of tachykinins and capsaicin on guinea-pig bronchial parasympathetic ganglion neurones

1. We evaluated the effects of neurokinins, tachykinin analogues, or capsaicin on passive membrane properties of guinea‐pig bronchial parasympathetic neurones using intracellular recording techniques. 2. Substance P (SP) and the tachykinin analogue, acetyl‐[Arg6,Sar9,Met(O2)11]‐SP(6‐11) (ASMSP), at concentrations selective for the neurokinin (NK)‐1 receptor subtype, depolarized the resting potential (3 and 5 mV, respectively) with no change in input resistance. Neurokinin A and beta Ala8NKA(4‐10), at concentrations selective for the NK‐2 receptor subtype (0.1 microM), were without effect. 3. Neurokinin B (NKB) and [Asp5,6,methyl‐Phe8]SP(5‐11) (senktide analogue), at concentrations selective for NK‐3 receptor subtype, elicited maximum depolarizations of 16 +/‐ 2 mV for both agonists. The peak of the depolarization was associated with an decrease in membrane resistance (35 +/‐ 4 and 50 +/‐ 7%, respectively). 4. Capsaicin (1 microM) elicited a 3‐24 mV depolarization of the resting potential of thirteen of eighteen bronchial ganglion neurones and decreased the input resistance of seven of thirteen of these neurones. The effects of capsaicin were reduced by desensitization with senktide analogue at a concentration selective for the NK‐3 receptor subtype, whereas a non‐peptide NK‐1 receptor antagonist had no effect. 5. Using voltage clamp analysis, capsaicin and senktide analogue evoked an inward current and an increase in membrane conductance at the resting membrane potential. The reversal potential for senktide analogue was estimated to be + 4 mV. 6. These data support the hypothesis that neurokinin‐containing nerve terminals are localized within guinea‐pig bronchial parasympathetic ganglia and, when released, the predominant effect of the neurokinins is by activation of NK‐3 receptors.

Heightened Endoplasmic Reticulum Stress in the Lungs of Patients with Chronic Obstructive Pulmonary Disease

RATIONALE Nuclear factor erythroid 2-related factor 2 (Nrf2), an important regulator of lung antioxidant defenses, declines in chronic obstructive pulmonary disease (COPD). However, Nrf2 also regulates the proteasome system that degrades damaged and misfolded proteins. Because accumulation of misfolded proteins in the endoplasmic reticulum (ER) causes ER stress and ER stress-induced apoptosis, Nrf2 may potentially prevent ER stress-mediated apoptosis in COPD. OBJECTIVES To determine whether Nrf2-regulated proteasome function affects ER stress-mediated apoptosis in COPD. METHODS We assessed the expression of Nrf2, Nrf2-dependent proteasomal subunits, proteasomal activity, markers of ER stress, and apoptosis in emphysematous lungs of mice exposed to cigarette smoke (CS) as well as peripheral lung tissues from normal control subjects and patients with COPD. MEASUREMENTS AND MAIN RESULTS Compared with wild-type mice, emphysematous lungs of CS-exposed Nrf2-deficient mice exhibited markedly lower proteasomal activity and elevated markers of ER stress and apoptosis. Furthermore, compared with normal control subjects, lungs of patients with mild and advanced COPD showed a marked decrease in the expression of Nrf2-regulated proteasomal subunits and total proteasomal activity. However, they were associated with greater levels of ER stress and apoptosis markers. In vitro studies have demonstrated that enhancing proteasomal activity in Beas2B cells either by sulforaphane, an activator of Nrf2, or overexpression of Nrf2-regulated proteasomal subunit PSMB6, significantly inhibited cigarette smoke condensate (CSC)-induced ER stress and cell death. CONCLUSIONS Impaired Nrf2 signaling causes significant decline in proteasomal activity and heightens ER stress response in lungs of patients with COPD and CS-exposed mice. Accordingly, pharmacological approaches that augment Nrf2 activity may protect against COPD progression by both up-regulating antioxidant defenses and relieving ER stress.

Chronic Intermittent Hypoxia Induces Atherosclerosis via Activation of Adipose Angiopoietin-like 4

RATIONALE Obstructive sleep apnea is a risk factor for dyslipidemia and atherosclerosis, which have been attributed to chronic intermittent hypoxia (CIH). Intermittent hypoxia inhibits a key enzyme of lipoprotein clearance, lipoprotein lipase, and up-regulates a lipoprotein lipase inhibitor, angiopoietin-like 4 (Angptl4), in adipose tissue. The effects and mechanisms of Angptl4 up-regulation in sleep apnea are unknown. OBJECTIVES To examine whether CIH induces dyslipidemia and atherosclerosis by increasing adipose Angptl4 via hypoxia-inducible factor-1 (HIF-1). METHODS ApoE(-/-) mice were exposed to intermittent hypoxia or air for 4 weeks while being treated with Angptl4-neutralizing antibody or vehicle. MEASUREMENTS AND MAIN RESULTS In vehicle-treated mice, hypoxia increased adipose Angptl4 levels, inhibited adipose lipoprotein lipase, increased fasting levels of plasma triglycerides and very low density lipoprotein cholesterol, and increased the size of atherosclerotic plaques. The effects of CIH were abolished by the antibody. Hypoxia-induced increases in plasma fasting triglycerides and adipose Angptl4 were not observed in mice with germline heterozygosity for a HIF-1α knockout allele. Transgenic overexpression of HIF-1α in adipose tissue led to dyslipidemia and increased levels of adipose Angptl4. In cultured adipocytes, constitutive expression of HIF-1α increased Angptl4 levels, which was abolished by siRNA. Finally, in obese patients undergoing bariatric surgery, the severity of nocturnal hypoxemia predicted Angptl4 levels in subcutaneous adipose tissue. CONCLUSIONS HIF-1-mediated increase in adipose Angptl4 and the ensuing lipoprotein lipase inactivation may contribute to atherosclerosis in patients with sleep apnea.

Cutting edge: Altered pulmonary eosinophilic inflammation in mice deficient for clara cell secretory 10-kDa protein

Clara cell secretory protein (CC10) is a steroid-inducible protein, and its in vivo function is currently unclear. The role of CC10 in modulation of pulmonary allergic inflammation was examined in mice deficient for the CC10 gene. Wild-type and homozygous CC10-deficient mice were sensitized with an Ag, OVA, and challenged with either OVA or saline. When compared with that seen in wild-type mice, a significantly higher level of pulmonary eosinophilia was found in Ag-sensitized and challenged CC10-deficient mice. Significantly increased levels of Th2 cytokines IL-4, IL-5, IL-9, and IL-13 were also found in CC10-deficient mice. In addition, an increased level of eotaxin, but not RANTES, was also seen in CC10-deficient mice. No significant difference was observed in the level of a Th1 cytokine, IFN-γ, between different groups of mice. These results provided the first in vivo evidence that CC10 plays a role in the modulation of pulmonary allergic inflammation.

Lysophosphatidic acid is detectable in human bronchoalveolar lavage fluids at baseline and increased after segmental allergen challenge

Background Lysophosphatidic acid (LPA) is a biologically active lysophospholipid and a component of normal plasma. LPA binds to receptors expressed on circulating and structural lung cells and affects cell growth and activation. Whether LPA is present in the lung has not been previously reported.