The organellar targeting of two-pore channels (TPCs) and their capacity to associate as homo- and heterodimers may be critical to endolysosomal signaling. A more detailed understanding of the ...functional association of vertebrate TPC1–3 is therefore necessary. We report here that when stably expressed in HEK293 cells, human (h) TPC1 and chicken (c) TPC3 were specifically targeted to different subpopulations of endosomes, hTPC2 was specifically targeted to lysosomes, and rabbit (r) TPC3 was specifically targeted to both endosomes and lysosomes. Intracellular dialysis of NAADP evoked a Ca2+ transient in HEK293 cells that stably overexpressed hTPC1, hTPC2, and rTPC3, but not in cells that stably expressed cTPC3. The Ca2+ transients induced in cells that overexpressed endosome-targeted hTPC1 were abolished upon depletion of acidic Ca2+ stores by bafilomycin A1, but remained unaffected following depletion of endoplasmic reticulum stores by thapsigargin. In contrast, Ca2+ transients induced via lysosome-targeted hTPC2 and endolysosome-targeted rTPC3 were abolished by bafilomycin A1 and markedly attenuated by thapsigargin. NAADP induced marked Ca2+ transients in HEK293 cells that stably coexpressed hTPC2 with hTPC1 or cTPC3, but failed to evoke any such response in cells that coexpressed interacting hTPC2 and rTPC3 subunits. We therefore conclude that 1) all three TPC subtypes may support Ca2+ signaling from their designate acidic stores, and 2) lysosome-targeted (but not endosome-targeted) TPCs support coupling to the endoplasmic reticulum.The role of two-pore channels (TPCs) in endolysosomal signaling remains controversial.
TPCs are targeted to different subpopulations of endolysosomes, and this determines subunit interaction and Ca2+ signaling by NAADP.
All vertebrate TPC subtypes support Ca2+ signaling; lysosome-targeted, but not endosome-targeted, TPCs permit endoplasmic reticulum coupling.
Organellar targeting and interaction of TPCs are likely critical to endolysosomal signaling in health and disease.
Modulation of breathing by hypoxia accommodates variations in oxygen demand and supply during, for example, sleep and ascent to altitude, but the precise molecular mechanisms of this phenomenon ...remain controversial. Among the genes influenced by natural selection in high-altitude populations is one for the adenosine monophosphate-activated protein kinase (AMPK) α1-catalytic subunit, which governs cell-autonomous adaptations during metabolic stress.
We investigated whether AMPK-α1 and/or AMPK-α2 are required for the hypoxic ventilatory response and the mechanism of ventilatory dysfunctions arising from AMPK deficiency.
We used plethysmography, electrophysiology, functional magnetic resonance imaging, and immediate early gene (c-fos) expression to assess the hypoxic ventilatory response of mice with conditional deletion of the AMPK-α1 and/or AMPK-α2 genes in catecholaminergic cells, which compose the hypoxia-responsive respiratory network from carotid body to brainstem.
AMPK-α1 and AMPK-α2 deletion virtually abolished the hypoxic ventilatory response, and ventilatory depression during hypoxia was exacerbated under anesthesia. Rather than hyperventilating, mice lacking AMPK-α1 and AMPK-α2 exhibited hypoventilation and apnea during hypoxia, with the primary precipitant being loss of AMPK-α1 expression. However, the carotid bodies of AMPK-knockout mice remained exquisitely sensitive to hypoxia, contrary to the view that the hypoxic ventilatory response is determined solely by increased carotid body afferent input to the brainstem. Regardless, functional magnetic resonance imaging and c-fos expression revealed reduced activation by hypoxia of well-defined dorsal and ventral brainstem nuclei.
AMPK is required to coordinate the activation by hypoxia of brainstem respiratory networks, and deficiencies in AMPK expression precipitate hypoventilation and apnea, even when carotid body afferent input is normal.
Two-pore segment channel 2 (TPC2) is a ubiquitously expressed, lysosomally targeted ion channel that aids in terminating autophagy and is inhibited upon its association with mechanistic target of ...rapamycin (mTOR). It is controversial whether TPC2 mediates lysosomal Ca
release or selectively conducts Na
and whether the binding of nicotinic acid adenine dinucleotide phosphate (NAADP) or phosphatidylinositol 3,5-bisphosphate PI(3,5)P
is required for the activity of this ion channel. We show that TPC2 is required for intracellular Ca
signaling in response to NAADP or to mTOR inhibition by rapamycin. In pulmonary arterial myocytes, rapamycin and NAADP evoked global Ca
transients that were blocked by depletion of lysosomal Ca
stores. Preincubation of cells with high concentrations of rapamycin resulted in desensitization and blocked NAADP-evoked Ca
signals. Moreover, rapamycin and NAADP did not evoke discernable Ca
transients in myocytes derived from
knockout mice, which showed normal responses to other Ca
-mobilizing signals. In HEK293 cells stably overexpressing human TPC2, shRNA-mediated knockdown of mTOR blocked rapamycin- and NAADP-evoked Ca
signals. Confocal imaging of a genetically encoded Ca
indicator fused to TPC2 demonstrated that rapamycin-evoked Ca
signals localized to lysosomes and were in close proximity to TPC2. Therefore, inactivation of mTOR may activate TPC2 and consequently lysosomal Ca
release.
Perhaps the defining characteristic of pulmonary arteries is the process of hypoxic pulmonary vasoconstriction (HPV) which, under physiological conditions, supports ventilation-perfusion matching in ...the lung by diverting blood flow away from oxygen deprived areas of the lung to oxygen rich regions. However, when alveolar hypoxia is more widespread, either at altitude or with disease (e.g., cystic fibrosis), HPV may lead to hypoxic pulmonary hypertension. HPV is driven by the intrinsic response to hypoxia of pulmonary arterial smooth muscle and endothelial cells, which are acutely sensitive to relatively small changes in pO2 and have evolved to monitor oxygen supply and thus address ventilation-perfusion mismatch. There is now a consensus that the inhibition by hypoxia of mitochondrial oxidative phosphorylation represents a key step towards the induction of HPV, but the precise nature of the signalling pathway(s) engaged thereafter remains open to debate. We will consider the role of the AMP-activated protein kinase (AMPK) and liver kinase B1 (LKB1), an upstream kinase through which AMPK is intimately coupled to changes in oxygen supply via mitochondrial metabolism. A growing body of evidence, from our laboratory and others, suggests that modulation of the LKB1-AMPK signalling pathway underpins both hypoxic pulmonary vasoconstriction and the development of pulmonary hypertension.
The microbial activities and the biodegradation-abilities of undefined consortium in contaminated soils in the Niger Delta of Nigeria were studied. The Respirametry technique was adopted to evaluate ...the microbial activities while the soils were incubated with 2% ( nu / nu ) crude oil in mineral salt medium at 37 degree C in three stages of two weeks each in a shake flask. At the end of the last phase, components of the crude oil degraded by the undefined consortium in the soils were identified with the gas chromatographic techniques. The consortia of the different samples studied showed different degree of capacities on the crude oil, removing a large number of components of the crude oil, making the areas potentially suitable for in-situ bioremediation.
To explore caregivers’ perceptions of childhood injuries in the rural and urban areas of India, with a focus on causes, consequences, prevention, and treatment. We conducted eight focus group ...discussions with fifty female caregivers in rural and urban areas of Ujjain in Central India and used thematic content analysis. The caregivers identified how children injured themselves through falls, road traffic injuries, metallic nails and tool injuries, ingestions of foreign objects and poisons, burns, drowning, and suffocation. The reported consequences of injuries ranged from pain, infections, scar formation, phobia, stigma, and emotional stress to complications like physical disability, loss of eyesight, head injury, paralysis, and even death. Many caregivers blamed children and their mischievousness for the injuries and failed to realise/acknowledge the role of better supervision and environmental modifications in injury prevention. Caregivers used several first aid methods to respond to injuries. These included applying pressure to stop bleeding during fall and road traffic injuries, inducing vomiting by giving the poison victims saltwater to drink, and tobacco leaves to chew. In addition, some caregivers resorted to using coconut oil and toothpaste on burnt skin and giving back blows for choking. Caregivers in communities had experiences of different types of child injuries. Further education on need for better supervision, relevant environmental modification and appropriate first aid treatment of various injuries is required.
The endoplasmic reticulum is not the only major agonist-releasable Ca2+ store within cells; it is now clear that virtually all organelles so far studied have the ability to act as mobilizable Ca2+ ...stores. From recent findings with regard to Ca2+ transportation and Ca2+ homeostasis within a variety of cell organelles such as the mitochondria, nucleus, Golgi and lysosomes, it emerges that many of these organellar Ca2+ stores appear to interact with each other, adding a further level of complexity to Ca2+ signalling events.
Tetrabromobisphenol A (TBBPA) is one of the most widely used members of the family of brominated flame retardants (BFRs). BFRs, including TBBPA have been shown to be widely distributed within the ...environment and there is growing evidence of their bio-accumulation within animals and man. Toxicological studies have shown that TBBPA can be harmful to cells by modulating a number of cell signalling processes. In this study, we employed fluorescence spectroscopy and differential scanning calorimetry to investigate the interaction of TBBPA with phospholipid membranes, as this is the most likely route for it to influence membrane-associated cellular processes. TBBPA readily and randomly partitions throughout all regions of the phospholipid bilayer with high efficacy {partition coefficient (Log
K
p)
=
5.7
±
0.7}. A decrease in membrane fluidity in both liquid-crystalline and gel-phase membranes was detected at concentrations of TBBPA as low as 2.5 μM. TBBPA also decreases the phase transition temperature of dipalmitoyl phoshatidylcholine (DPPC) membranes and broadened transition peaks, in a fashion similar to that for cholesterol. TBBPA, however, also prefers to partition into membrane regions not too highly enriched with cholesterol. Our findings therefore suggests that, the toxic effects of TBBPA, may at least in part, be due to its lipid membrane binding/perturbing effects, which in turn, could influence biological processes involving cell membranes.
Alkylphenols such as nonylphenol are pollutants that are widely dispersed within our environment. They bio-accumulate within man, with levels in the muM concentration range reported in human tissues. ...These chemicals act as endocrine disruptors, having xenoestrogenic activity. More recently alkylphenols have also been shown to affect Ca2+ signalling pathways. Here we show that alkylphenols are potent inhibitors of sarcoplasmic-endoplasmic reticulum Ca2+-ATPase (SERCA) activity. For linear chain alkylphenols the potency of inhibition is related to chain length, with the IC50 values for inhibition ranging from 8 microM for 4-n-nonylphenol (C9) to 1.3 mM for 4-n-propylphenol (C3). Branched chain alkylphenols generally had lower potencies than their linear chain counterparts, however, good correlations for all alkylphenols were observed between their Ca2+ pump inhibition and hydrophobicity, molecular volume and flexibility, indicating that these parameters are all important factors. Alkylphenols cause abnormal elevations of intracellular Ca2+ within TM4 Sertoli cells (cells involved in sperm maturation) depolarise their mitochondria and induce cell death in these cells, in an alkyl chain size-dependent manner.
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