Josef Buttigieg

Neurotransmitter mechanisms mediating low-glucose signalling in cocultures and fresh tissue slices of rat carotid body.

The mammalian carotid body (CB) is a polymodal chemosensor which can detect low blood glucose (hypoglycaemia), leading to increased afferent discharge and activation of counter‐regulatory autonomic pathways. The underlying neurotransmitter mechanisms are unknown and controversy surrounds whether the action of low glucose is direct or indirect. To address this, we used a coculture model containing functional chemosensory units of rat CB receptor (type I) cell clusters and afferent petrosal neurones (PN). During perforated‐patch, whole‐cell recordings, low glucose (0–2 mm) stimulated sensory discharge in cocultured PN. When the background P  O 2 was lowered to levels typical of arterial blood (∼90 mmHg), robust PN chemoexcitation could be induced by physiological hypoglycaemia (3.3–4 mm glucose). These sensory responses were reversibly inhibited by a combination of purinergic (suramin, 50 μm) and nicotinic (mecamylamine, 1 μm) receptor blockers, suggesting that transmission depended on corelease of ATP and ACh. Hypoglycaemic responses were additive with those evoked by hypoxia or hypercapnia; further, they could be potentiated by the GABAB receptor blocker (CGP 55845) and inhibited by 5‐HT2A receptor blockers (ketanserin or ritanserin). During paired simultaneous recordings from a PN and a type I cell in an adjacent cluster, the afferent PN response coincided with type I cell depolarization, which was associated with a decrease in input resistance. In fresh tissue slices of rat CB, low glucose stimulated ATP secretion as determined by the luciferin–luciferase assay; this secretion was cadmium sensitive, potentiated by CGP 55845, and inhibited by ketanserin. Taken together these data indicate that CB receptors act as direct glucosensors, and that processing of hypoglycaemia utilizes similar neurotransmitter and neuromodulatory mechanisms as hypoxia.

Delayed Post-Injury Administration of Riluzole Is Neuroprotective in a Preclinical Rodent Model of Cervical Spinal Cord Injury

Riluzole, a sodium/glutamate antagonist has shown promise as a neuroprotective agent. It is licensed for amyotrophic lateral sclerosis and is in clinical trial development for spinal cord injury (SCI). This study investigated the therapeutic time-window and pharmacokinetics of riluzole in a rodent model of cervical SCI. Rats were treated with riluzole (8 mg/kg) at 1 hour (P1) and 3 hours (P3) after injury or with vehicle. Afterward, P1 and P3 groups received riluzole (6 (mg/kg) every 12 hours for 7 days. Both P1 and P3 animals had significant improvements in locomotor recovery as measured by open field locomotion (BBB score, BBB subscore). Von Frey stimuli did not reveal an increase in at level or below level mechanical allodynia. Sensory-evoked potential recordings and quantification of axonal cytoskeleton demonstrated a riluzole-mediated improvement in axonal integrity and function. Histopathological and retrograde tracing studies demonstrated that delayed administration leads to tissue preservation and reduces apoptosis and inflammation. High performance liquid chromatography (HPLC) was undertaken to examine the pharmacokinetics of riluzole. Riluzole penetrates the spinal cord in 15 min, and SCI slowed elimination of riluzole from the spinal cord, resulting in a longer half-life and higher drug concentration in spinal cord and plasma. Initiation of riluzole treatment 1 and 3 hours post-SCI led to functional, histological, and molecular benefits. While extrapolation of post-injury time windows from rat to man is challenging, evidence from SCI-related biomarker studies would suggest that the post-injury time window is likely to be at least 12 hours in man.

A rotenone-sensitive site and H2O2 are key components of hypoxia-sensing in neonatal rat adrenomedullary chromaffin cells

In the perinatal period, adrenomedullary chromaffin cells (AMC) directly sense PO2 and secrete catecholamines during hypoxic stress, and this response is lost in juvenile ( approximately 2 week-old) chromaffin cells following postnatal innervation. Here we tested the hypothesis that a rotenone-sensitive O2-sensor and ROS are involved in the hypoxic response of AMC cultured from neonatal and juvenile rats. In whole-cell recordings, hypoxia (PO2=5-15 mm Hg) inhibited outward current in neonatal AMC; this response was reversed by exogenous H2O2 and mimicked and occluded by intracellular catalase (1000 units/ml), as well as the antioxidants, N-acetyl-L-cysteine (NAC; 50 microM) and Trolox (200 microM). Acute hypoxia decreased ROS levels and stimulated ATP secretion in these cells, as measured by luminol and luciferin-luciferase chemiluminescence, respectively. Of several mitochondrial electron transport chain (ETC) inhibitors tested, only rotenone, a complex I blocker, mimicked and occluded the effects of hypoxia on outward current, cellular ROS, and ATP secretion. Succinate donors, which act as complex II substrates, reversed the effects of hypoxia and rotenone in neonatal AMC. In contrast, in hypoxia-insensitive juvenile AMC, neither NAC nor rotenone stimulated ATP secretion though they both caused a decrease in ROS levels. We propose that O2-sensing by neonatal AMC is mediated by decreased ROS generation via a rotenone-sensitive site that is coupled to outward current inhibition and secretion. Interestingly, juvenile AMC display at least two modifications, i.e. an uncoupling of the O2-sensor from ROS regulation, and an apparent insensitivity of outward current to decreased ROS.

Functional mitochondria are required for O2 but not CO2 sensing in immortalized adrenomedullary chromaffin cells

Catecholamine (CAT) release from adrenomedullary chromaffin cells (AMC) in response to stressors such as low O(2) (hypoxia) and elevated CO(2)/H(+) is critical during adaptation of the newborn to extrauterine life. Using a surrogate model based on a v-myc immortalized adrenal chromaffin cell line (i.e., MAH cells), combined with genetic perturbation of mitochondrial function, we tested the hypothesis that functional mitochondria are required for O(2) sensing. Wild-type MAH cells responded to both hypoxia and increased CO(2) (hypercapnia) with K(+) current inhibition and membrane depolarization. Additionally, these stimuli caused a rise in cytosolic Ca(2+) and CAT secretion, determined by fura-2 spectrofluorimetry and carbon fiber amperometry, respectively. In contrast, mitochondria-deficient (rho(0)) MAH cells were hypoxia insensitive, although responses to hypercapnia and expression of several markers, including carbonic anhydrase II, remained intact. Rotenone (1 microM), a mitochondrial complex I blocker known to mimic and occlude the effects of hypoxia in primary AMC, was effective in wild-type but not rho(0) MAH cells. These data demonstrate that functional mitochondria are involved in hypoxia-sensing by adrenal chromaffin cells. We also show for the first time that, like their neonatal chromaffin cell counterparts, MAH cells are CO(2) sensors; however, this property is independent of functional mitochondria.

The Generation of Definitive Neural Stem Cells from PiggyBac Transposon-Induced Pluripotent Stem Cells Can Be Enhanced by Induction of the NOTCH Signaling Pathway

Cell-based therapies using neural stem cells (NSCs) have shown positive outcomes in various models of neurological injury and disease. Induced pluripotent stem cells (iPSCs) address many problems associated with NSCs from various sources, including the immune response and cell availability. However, due to inherent differences between embryonic stem cells (ESCs) and iPSCs, detailed characterization of the iPS-derived NSCs will be required before translational experiments can be performed. Murine piggyBac transposon iPSCs were clonally expanded in floating sphere colonies to generate primitive NSCs initially with serum-free media (SFM) containing the leukemia inhibitory factor and followed by SFM with the fibroblast growth factor-2 (FGF2) to form colonies of definitive NSCs (dNSCs). Primitive and definitive clonally derived neurospheres were successfully generated using the default conditions from iPSCs and ESCs. However, the iPSC-dNSCs expressed significantly higher levels of pluripotency and nonectoderm lineage genes compared to equivalent ESC-dNSCs. The addition of the bone morphogenetic proteins antagonist, Noggin, to the media significantly increased primary neurosphere generation from the iPSC lines, but did not affect the dNSC sphere colonies generated. The induction of the NOTCH pathway by the Delta-like ligand 4 (DLL4) improved the generation and quality of dNSCs, as demonstrated by a reduction in pluripotency and nonectodermal markers, while maintaining NSC-specific gene expression. The iPS-dNSCs (+DLL4) showed functional neural differentiation by immuncytochemical staining and electrophysiology. This study suggests the intrinsic differences between ESCs and iPSCs in their ability to acquire a dNSC fate that can be overcome by inducing the NOTCH pathway.

Chronic nicotine in utero selectively suppresses hypoxic sensitivity in neonatal rat adrenal chromaffin cells

Nicotine in cigarette smoke has been linked to several deleterious side effects on the off spring of smoking mothers, including impaired devel opment of the sympathoadrenal system, abnormal arousal reflexes, and sudden infant death syndrome. Catecholamine (CA) release from adrenomedullary chromaffin cells (AMCs) in response to asphyxial stres sors, e.g., low O2 (hypoxia) and elevated CO2 (hyper capnia), is critical for adaptation to extrauterine life and occurs before splanchnic innervation. Here, we investigated the effects of prenatal nicotine bitartrate exposure on the ability of neonatal (P0) rat AMCs to respond appropriately to asphyxial stressors. Control AMCs isolated from pups born to saline‐treated dams displayed typical responses to hypoxia and hypercap nia, including inhibition of outward K+ current, mem brane depolarization, increased cytosolic calcium, and CA secretion. In contrast, P0 AMCs from pups born to nicotine‐treated dams showed a marked suppression or loss of hypoxic sensitivity, although hypercapnic sensi tivity and the expression of CO2 markers (i.e., carbonic anhydrase I and II) appeared normal. Moreover, iso lated saline‐treated P0 AMCs lost their hypoxic sensi tivity when grown in culture for ~ 1 wk in the presence of a subsaturating concentration of nicotine base (50 μM), and this effect was abolished by the nicotinic acetylcholine receptor (nAChR) blocker mecamylamine (100 μM). Taken together, these data suggest that the adverse effects of maternal smoking on sympathoadre nal function in the offspring are due in part to a loss or suppression of acute hypoxic sensitivity in adrenal chromaffin cells, triggered by the direct action of nicotine on endogenous nicotinic acetylcholine receptors.—Buttigieg, J., Brown, S., Zhang, M., Lowe, M., Holloway, A. C., Nurse, C. A. Chronic nicotine in utero selectively suppresses hypoxic sensitivity in neonatal rat adrenal chromaffin cells. FASEB J. 22, 1317–1326 (2008)

Chronic Nicotine Blunts Hypoxic Sensitivity in Perinatal Rat Adrenal Chromaffin Cells via Upregulation of KATP Channels: Role of α7 Nicotinic Acetylcholine Receptor and Hypoxia-Inducible Factor-2α

Fetal nicotine exposure blunts hypoxia-induced catecholamine secretion from neonatal adrenomedullary chromaffin cells (AMCs), providing a link between maternal smoking, abnormal arousal responses, and risk of sudden infant death syndrome. Here, we show that the mechanism is attributable to upregulation of KATP channels via stimulation of α7 nicotinic ACh receptors (AChRs). These KATP channels open during hypoxia, thereby suppressing membrane excitability. After in utero exposure to chronic nicotine, neonatal AMCs show a blunted hypoxic sensitivity as determined by inhibition of outward K+ current, membrane depolarization, rise in cytosolic Ca2+, and catecholamine secretion. However, hypoxic sensitivity could be unmasked in nicotine-exposed AMCs when glibenclamide, a blocker of KATP channels, was present. Both KATP current density and KATP channel subunit (Kir 6.2) expression were significantly enhanced in nicotine-exposed cells relative to controls. The entire sequence could be reproduced in culture by exposing neonatal rat AMCs or immortalized fetal chromaffin (MAH) cells to nicotine for ∼1 week, and was prevented by coincubation with selective blockers of α7 nicotinic AChRs. Additionally, coincubation with inhibitors of protein kinase C and CaM kinase, but not protein kinase A, prevented the effects of chronic nicotine in vitro. Interestingly, chronic nicotine failed to blunt hypoxia-evoked responses in MAH cells bearing short hairpin knockdown (>90%) of the transcription factor, hypoxia-inducible factor-2α (HIF-2α), suggesting involvement of the HIF pathway. The therapeutic potential of KATP channel blockers was validated in experiments in which hypoxia-induced neonatal mortality in nicotine-exposed pups was significantly reduced after pretreatment with glibenclamide.

Regulation of oxygen sensitivity in adrenal chromaffin cells.

Adrenomedullary chromaffin cells (AMC) possess a direct hypoxia‐sensing mechanism that promotes a vital catecholamine surge at birth. This mitochondria‐dependent adaptive mechanism is suppressed postnatally as AMC acquire cholinergic innervation, and it is mediated by K+ channel inhibition, membrane depolarization, and voltage‐gated Ca2+ entry. We hypothesized that nicotinic ACh receptor (AChR) activation might contribute to this postnatal loss of O2 sensitivity. Following in utero nicotinic AChR activation, via maternal administration of nicotine bitartrate, hypoxic sensitivity was suppressed in neonatal AMC. Similarly, when neonatal AMC or immortalized chromaffin (MAH) cells were cultured for ∼7 d with nicotine base (50 μM), hypoxic sensitivity was suppressed. This effect required α7 nAChR stimulation, and involved upregulation of KATP channels, which are activated during hypoxia. Thus, nicotinic AChR activation may contribute to the suppression of hypoxic sensitivity in AMC, and this pathway could provide the basis for the loss of hypoxia tolerance in the offspring of smoking mothers.

NOX2 (gp91phox) is a predominant O2 sensor in a human airway chemoreceptor cell line: biochemical, molecular, and electrophysiological evidence

Pulmonary neuroepithelial bodies (NEBs), composed of clusters of amine [serotonin (5-HT)] and peptide-producing cells, are widely distributed within the airway mucosa of human and animal lungs. NEBs are thought to function as airway O(2)-sensors, since they are extensively innervated and release 5-HT upon hypoxia exposure. The small cell lung carcinoma cell line (H146) provides a useful model for native NEBs, since they contain (and secrete) 5-HT and share the expression of a membrane-delimited O(2) sensor [classical NADPH oxidase (NOX2) coupled to an O(2)-sensitive K(+) channel]. In addition, both native NEBs and H146 cells express different NADPH oxidase homologs (NOX1, NOX4) and its subunits together with a variety of O(2)-sensitive voltage-dependent K(+) channel proteins (K(v)) and tandem pore acid-sensing K(+) channels (TASK). Here we used H146 cells to investigate the role and interactions of various NADPH oxidase components in O(2)-sensing using a combination of coimmunoprecipitation, Western blot analysis (quantum dot labeling), and electrophysiology (patchclamp, amperometry) methods. Coimmunoprecipitation studies demonstrated formation of molecular complexes between NOX2 and K(v)3.3 and K(v)4.3 ion channels but not with TASK1 ion channels, while NOX4 associated with TASK1 but not with K(v) channel proteins. Downregulation of mRNA for NOX2, but not for NOX4, suppressed hypoxia-sensitive outward current and significantly reduced hypoxia -induced 5-HT release. Collectively, our studies suggest that NOX2/K(v) complexes are the predominant O(2) sensor in H146 cells and, by inference, in native NEBs. Present findings favor a NEB cell-specific plasma membrane model of O(2)-sensing and suggest that unique NOX/K(+) channel combinations may serve diverse physiological functions.

PpASCL, the Physcomitrella patens Anther-Specific Chalcone Synthase-Like Enzyme Implicated in Sporopollenin Biosynthesis, Is Needed for Integrity of the Moss Spore Wall and Spore Viability.

Sporopollenin is the main constituent of the exine layer of spore and pollen walls. The anther-specific chalcone synthase-like (ASCL) enzyme of Physcomitrella patens, PpASCL, has previously been implicated in the biosynthesis of sporopollenin, the main constituent of exine and perine, the two outermost layers of the moss spore cell wall. We made targeted knockouts of the corresponding gene, PpASCL, and phenotypically characterized ascl sporophytes and spores at different developmental stages. Ascl plants developed normally until late in sporophytic development, when the spores produced were structurally aberrant and inviable. The development of the ascl spore cell wall appeared to be arrested early in microspore development, resulting in small, collapsed spores with altered surface morphology. The typical stratification of the spore cell wall was absent with only an abnormal perine recognisable above an amorphous layer possibly representing remnants of compromised intine and/or exine. Equivalent resistance of the spore walls of ascl mutants and the control strain to acetolysis suggests the presence of chemically inert, defective sporopollenin in the mutants. Anatomical abnormalities of late-stage ascl sporophytes include a persistent large columella and an air space incompletely filled with spores. Our results indicate that the evolutionarily conserved PpASCL gene is needed for proper construction of the spore wall and for normal maturation and viability of moss spores.