Getting sweeter: new evidence for glucose transporters in specific cell types of the airway?

New technologies such as single-cell RNA sequencing (scRNAseq) has enabled identification of the mRNA transcripts expressed by individual cells. This review provides insight from recent scRNAseq studies on the expression of glucose transporters in the epithelial cells of the airway epithelium from trachea to alveolus. The number of studies analyzed was limited, not all reported the full range of glucose transporters and there were differences between cells freshly isolated from the airways and those grown in vitro. Furthermore, glucose transporter mRNA transcripts were expressed at lower levels than other epithelial marker genes. Nevertheless, these studies highlighted that there were differences in cellular expression of glucose transporters. GLUT1 was the most abundant of the broadly expressed transporters that included GLUT8, 10, and 13. GLUT9 transcripts were more common in basal cells and GLUT12 in ionocytes/ciliated cells. In addition to alveolar cells, SGLT1 transcripts were present in secretory cells. GLUT3 mRNA transcripts were expressed in a cell cluster that expressed monocarboxylate (MCT2) transporters. Such distributions likely underlie cell-specific metabolic requirements to support proliferation, ion transport, mucous secretion, environment sensing, and airway glucose homeostasis. These studies have also highlighted the role of glucose transporters in the movement of dehydroascorbic acid/vitamin C/myoinositol/urate, which are factors important to the innate immune properties of the airways. Discrepancies remain between detection of mRNAs, protein, and function of glucose transporters in the lungs. However, collation of the data from further scRNAseq studies may provide a better consensus and understanding, supported by qPCR, immunohistochemistry, and functional experiments.

Metformin attenuates the effect of Staphylococcus aureus on airway tight junctions by increasing PKCζ-mediated phosphorylation of occludin.

Airway epithelial tight junction (TJ) proteins form a resistive barrier to the external environment, however, during respiratory bacterial infection TJs become disrupted compromising barrier function. This promotes glucose flux/accumulation into the lumen which acts as a nutrient source for bacterial growth. Metformin used for the treatment of diabetes increases transepithelial resistance (TEER) and partially prevents the effect of bacteria but the mechanisms of action are unclear. We investigated the effect of metformin and Staphylococcus aureus on TJ proteins, zonula occludins (ZO)-1 and occludin in human airway epithelial cells (H441). We also explored the role of AMP-activated protein kinase (AMPK) and PKCζ in metformin-induced effects. Pretreatment with metformin prevented the S. aureus-induced changes in ZO-1 and occludin. Metformin also promoted increased abundance of full length over smaller cleaved occludin proteins. The nonspecific PKC inhibitor staurosporine reduced TEER but did not prevent the effect of metformin indicating that the pathway may involve atypical PKC isoforms. Investigation of TJ reassembly after calcium depletion showed that metformin increased TEER more rapidly and promoted the abundance and localization of occludin at the TJ. These effects were inhibited by the AMPK inhibitor, compound C and the PKCζ pseudosubstrate inhibitor (PSI). Metformin increased phosphorylation of occludin and acetyl-coA-carboxylase but only the former was prevented by PSI. This study demonstrates that metformin improves TJ barrier function by promoting the abundance and assembly of full length occludin at the TJ and that this process involves phosphorylation of the protein via an AMPK-PKCζ pathway.

Mechanisms for the control of local tissue blood flow during thermal interventions: influence of temperature-dependent ATP release from human blood and endothelial cells.

NEW FINDINGS: What is the central question of this study? Skin and muscle blood flow increases with heating and decreases with cooling, but the temperature-sensitive mechanisms underlying these responses are not fully elucidated. What is the main finding and its importance? We found that local tissue hyperaemia was related to elevations in ATP release from erythrocytes. Increasing intravascular ATP augmented skin and tissue perfusion to levels equal or above thermal hyperaemia. ATP release from isolated erythrocytes was altered by heating and cooling. Our findings suggest that erythrocytes are involved in thermal regulation of blood flow via modulation of ATP release. Local tissue perfusion changes with alterations in temperature during heating and cooling, but the thermosensitivity of the vascular ATP signalling mechanisms for control of blood flow during thermal interventions remains unknown. Here, we tested the hypotheses that the release of the vasodilator mediator ATP from human erythrocytes, but not from endothelial cells or other blood constituents, is sensitive to both increases and reductions in temperature and that increasing intravascular ATP availability with ATP infusion would potentiate thermal hyperaemia in limb tissues. We first measured blood temperature, brachial artery blood flow and plasma [ATP] during passive arm heating and cooling in healthy men and found that they increased by 3.0 ± 1.2°C, 105 ± 25 ml min-1  °C-1 and twofold, respectively, (all P < 0.05) with heating, but decreased or remained unchanged with cooling. In additional men, infusion of ATP into the brachial artery increased skin and deep tissue perfusion to levels equal or above thermal hyperaemia. In isolated erythrocyte samples exposed to different temperatures, ATP release increased 1.9-fold from 33 to 39°C (P < 0.05) and declined by ∼50% at 20°C (P < 0.05), but no changes were observed in cultured human endothelial cells, plasma or serum samples. In conclusion, increases in plasma [ATP] and skin and deep tissue perfusion with limb heating are associated with elevations in ATP release from erythrocytes, but not from endothelial cells or other blood constituents. Erythrocyte ATP release is also sensitive to temperature reductions, suggesting that erythrocytes may function as thermal sensors and ATP signalling generators for control of tissue perfusion during thermal interventions.

Dehydration accelerates reductions in cerebral blood flow during prolonged exercise in the heat without compromising brain metabolism.

Dehydration hastens the decline in cerebral blood flow (CBF) during incremental exercise, whereas the cerebral metabolic rate for O2 (CMRO2 ) is preserved. It remains unknown whether CMRO2 is also maintained during prolonged exercise in the heat and whether an eventual decline in CBF is coupled to fatigue. Two studies were undertaken. In study 1, 10 male cyclists cycled in the heat for ∼2 h with (control) and without fluid replacement (dehydration) while internal and external carotid artery blood flow and core and blood temperature were obtained. Arterial and internal jugular venous blood samples were assessed with dehydration to evaluate CMRO2 . In study 2, in 8 male subjects, middle cerebral artery blood velocity was measured during prolonged exercise to exhaustion in both dehydrated and euhydrated states. After a rise at the onset of exercise, internal carotid artery flow declined to baseline with progressive dehydration (P < 0.05). However, cerebral metabolism remained stable through enhanced O2 and glucose extraction (P < 0.05). External carotid artery flow increased for 1 h but declined before exhaustion. Fluid ingestion maintained cerebral and extracranial perfusion throughout nonfatiguing exercise. During exhaustive exercise, however, euhydration delayed but did not prevent the decline in cerebral perfusion. In conclusion, during prolonged exercise in the heat, dehydration accelerates the decline in CBF without affecting CMRO2 and also restricts extracranial perfusion. Thus, fatigue is related to a reduction in CBF and extracranial perfusion rather than CMRO2 .

Local temperature-sensitive mechanisms are important mediators of limb tissue hyperemia in the heat-stressed human at rest and during small muscle mass exercise.

Limb tissue and systemic blood flow increases with heat stress, but the underlying mechanisms remain poorly understood. Here, we tested the hypothesis that heat stress-induced increases in limb tissue perfusion are primarily mediated by local temperature-sensitive mechanisms. Leg and systemic temperatures and hemodynamics were measured at rest and during incremental single-legged knee extensor exercise in 15 males exposed to 1 h of either systemic passive heat-stress with simultaneous cooling of a single leg (n = 8) or isolated leg heating or cooling (n = 7). Systemic heat stress increased core, skin and heated leg blood temperatures (Tb), cardiac output, and heated leg blood flow (LBF; 0.6 ± 0.1 l/min; P < 0.05). In the cooled leg, however, LBF remained unchanged throughout (P > 0.05). Increased heated leg deep tissue blood flow was closely related to Tb (R(2) = 0.50; P < 0.01), which is partly attributed to increases in tissue V̇O2 (R(2) = 0.55; P < 0.01) accompanying elevations in total leg glucose uptake (P < 0.05). During isolated limb heating and cooling, LBFs were equivalent to those found during systemic heat stress (P > 0.05), despite unchanged systemic temperatures and hemodynamics. During incremental exercise, heated LBF was consistently maintained ∼ 0.6 l/min higher than that in the cooled leg (P < 0.01), with LBF and vascular conductance in both legs showing a strong correlation with their respective local Tb (R(2) = 0.85 and 0.95, P < 0.05). We conclude that local temperature-sensitive mechanisms are important mediators in limb tissue perfusion regulation both at rest and during small-muscle mass exercise in hyperthermic humans.

Dehydration affects cerebral blood flow but not its metabolic rate for oxygen during maximal exercise in trained humans.

Intense exercise is associated with a reduction in cerebral blood flow (CBF), but regulation of CBF during strenuous exercise in the heat with dehydration is unclear. We assessed internal (ICA) and common carotid artery (CCA) haemodynamics (indicative of CBF and extra-cranial blood flow), middle cerebral artery velocity (MCA Vmean), arterial-venous differences and blood temperature in 10 trained males during incremental cycling to exhaustion in the heat (35°C) in control, dehydrated and rehydrated states. Dehydration reduced body mass (75.8 ± 3 vs. 78.2 ± 3 kg), increased internal temperature (38.3 ± 0.1 vs. 36.8 ± 0.1°C), impaired exercise capacity (269 ± 11 vs. 336 ± 14 W), and lowered ICA and MCA Vmean by 12-23% without compromising CCA blood flow. During euhydrated incremental exercise on a separate day, however, exercise capacity and ICA, MCA Vmean and CCA dynamics were preserved. The fast decline in cerebral perfusion with dehydration was accompanied by increased O2 extraction (P < 0.05), resulting in a maintained cerebral metabolic rate for oxygen (CMRO2). In all conditions, reductions in ICA and MCA Vmean were associated with declining cerebral vascular conductance, increasing jugular venous noradrenaline, and falling arterial carbon dioxide tension (P aCO 2) (R(2) ≥ 0.41, P ≤ 0.01) whereas CCA flow and conductance were related to elevated blood temperature. In conclusion, dehydration accelerated the decline in CBF by decreasing P aCO 2 and enhancing vasoconstrictor activity. However, the circulatory strain on the human brain during maximal exercise does not compromise CMRO2 because of compensatory increases in O2 extraction.

Proinflammatory mediators disrupt glucose homeostasis in airway surface liquid.

The glucose concentration of the airway surface liquid (ASL) is much lower than that in blood and is tightly regulated by the airway epithelium. ASL glucose is elevated in patients with viral colds, cystic fibrosis, chronic obstructive pulmonary disease, and asthma. Elevated ASL glucose is also associated with increased incidence of respiratory infection. However, the mechanism by which ASL glucose increases under inflammatory conditions is unknown. The aim of this study was to investigate the effect of proinflammatory mediators (PIMs) on the mechanisms governing airway glucose homeostasis in polarized monolayers of human airway (H441) and primary human bronchial epithelial (HBE) cells. Monolayers were treated with TNF-α, IFN-γ, and LPS during 72 h. PIM treatment led to increase in ASL glucose concentration and significantly reduced H441 and HBE transepithelial resistance. This decline in transepithelial resistance was associated with an increase in paracellular permeability of glucose. Similar enhanced rates of paracellular glucose flux were also observed across excised trachea from LPS-treated mice. Interestingly, PIMs enhanced glucose uptake across the apical, but not the basolateral, membrane of H441 and HBE monolayers. This increase was predominantly via phloretin-sensitive glucose transporter (GLUT)-mediated uptake, which coincided with an increase in GLUT-2 and GLUT-10 abundance. In conclusion, exposure of airway epithelial monolayers to PIMs results in increased paracellular glucose flux, as well as apical GLUT-mediated glucose uptake. However, uptake was insufficient to limit glucose accumulation in ASL. To our knowledge, these data provide for the first time a mechanism to support clinical findings that ASL glucose concentration is increased in patients with airway inflammation.

Haemodynamic responses to dehydration in the resting and exercising human leg.

Dehydration and hyperthermia reduces leg blood flow (LBF), cardiac output ([Formula: see text]) and arterial pressure during whole-body exercise. It is unknown whether the reductions in blood flow are associated with dehydration-induced alterations in arterial blood oxygen content (C aO2) and O2-dependent signalling. This study investigated the impact of dehydration and concomitant alterations in C aO2 upon LBF and [Formula: see text]. Haemodynamics, arterial and femoral venous blood parameters and plasma [ATP] were measured at rest and during one-legged knee-extensor exercise in 7 males in four conditions: (1) control, (2) mild dehydration, (3) moderate dehydration, and (4) rehydration. Relative to control, C aO2 and LBF increased with dehydration at rest and during exercise (C aO2: from 199 ± 1 to 208 ± 2, and 202 ± 2 to 210 ± 2 ml L(-1) and LBF: from 0.38 ± 0.04 to 0.77 ± 0.09, and 1.64 ± 0.09 to 1.88 ± 0.1 L min(-1), respectively). Similarly, [Formula: see text] was unchanged or increased with dehydration at rest and during exercise, whereas arterial and leg perfusion pressures declined. Following rehydration, C aO2 declined (to 193 ± 2 mL L(-1)) but LBF remained elevated. Alterations in LBF were unrelated to C aO2 (r (2) = 0.13-0.27, P = 0.48-0.64) and plasma [ATP]. These findings suggest dehydration and concomitant alterations in C aO2 do not compromise LBF despite reductions in plasma [ATP]. While an additive or synergistic effect cannot be excluded, reductions in LBF during exercise with dehydration may not necessarily be associated with alterations in C aO2 and/or intravascular [ATP].

Lipoxin receptor agonist and inhibition of LTA4 hydrolase prevent tight junction disruption caused by P. aeruginosa filtrate in airway epithelial cells.

Airway diseases can disrupt tight junction proteins, compromising the epithelial barrier and making it more permeable to pathogens. In people with pulmonary disease who are prone to infection with Pseudomonas aeruginosa, pro-inflammatory leukotrienes are increased and anti-inflammatory lipoxins are decreased. Upregulation of lipoxins is effective in counteracting inflammation and infection. However, whether combining a lipoxin receptor agonist with a specific leukotriene A4 hydrolase (LTA4H) inhibitor could enhance these protective effects has not to our knowledge been investigated. Therefore, we explored the effect of lipoxin receptor agonist BML-111 and JNJ26993135 a specific LTA4H inhibitor that prevents the production of pro-inflammatory LTB4 on tight junction proteins disrupted by P. aeruginosa filtrate (PAF) in human airway epithelial cell lines H441 and 16HBE-14o. Pre-treatment with BML-111 prevented an increase in epithelial permeability induced by PAF and conserved ZO-1 and claudin-1 at the cell junctions. JNJ26993135 similarly prevented the increased permeability induced by PAF, restored ZO-1 and E-cadherin and reduced IL-8 but not IL-6. Cells pre-treated with BML-111 plus JNJ26993135 restored TEER and permeability, ZO-1 and claudin-1 to the cell junctions. Taken together, these data indicate that the combination of a lipoxin receptor agonist with a LTA4H inhibitor could provide a more potent therapy.

Temperature-dependent release of ATP from human erythrocytes: mechanism for the control of local tissue perfusion.

Human limb muscle and skin blood flow increases significantly with elevations in temperature, possibly through physiological processes that involve temperature-sensitive regulatory mechanisms. Here we tested the hypothesis that the release of the vasodilator ATP from human erythrocytes is sensitive to physiological increases in temperature both in vitro and in vivo, and examined potential channel/transporters involved. To investigate the source of ATP release, whole blood, red blood cells (RBCs), plasma and serum were heated in vitro to 33, 36, 39 and 42°C. In vitro heating augmented plasma or 'bathing solution' ATP in whole blood and RBC samples, but not in either isolated plasma or serum samples. Heat-induced ATP release was blocked by niflumic acid and glibenclamide, but was not affected by inhibitors of nucleoside transport or anion exchange. Heating blood to 42°C enhanced (P < 0.05) membrane protein abundance of cystic fibrosis transmembrane conductance regulator (CFTR) in RBCs. In a parallel in vivo study in humans exposed to whole-body heating at rest and during exercise, increases in muscle temperature from 35 to 40°C correlated strongly with elevations in arterial plasma ATP (r(2) = 0.91; P = 0.0001), but not with femoral venous plasma ATP (r(2) = 0.61; P = 0.14). In vitro, however, the increase in ATP release from RBCs was similar in arterial and venous samples heated to 39°C. Our findings demonstrate that erythrocyte ATP release is sensitive to physiological increases in temperature, possibly via activation of CFTR-like channels, and suggest that temperature-dependent release of ATP from erythrocytes might be an important mechanism regulating human limb muscle and skin perfusion in conditions that alter blood and tissue temperature.

Glucose homeostasis across human airway epithelial cell monolayers: role of diffusion, transport and metabolism.

Glucose in airway surface liquid (ASL) is maintained at low concentrations compared to blood glucose. Using radiolabelled [(3)H]-D: -glucose and [(14)C]-L: -glucose, detection of D: - and L: -glucose by high-performance liquid chromatography and metabolites by nuclear magnetic resonance, we found that glucose applied to the basolateral side of H441 human airway epithelial cell monolayers at a physiological concentration (5 mM) crossed to the apical side by paracellular diffusion. Transepithelial resistance of the monolayer was inversely correlated with paracellular diffusion. Appearance of glucose in the apical compartment was reduced by uptake of glucose into the cell by basolateral and apical phloretin-sensitive GLUT transporters. Glucose taken up into the cell was metabolised to lactate which was then released, at least in part, across the apical membrane. We suggest that glucose transport through GLUT transporters and its subsequent metabolism in lung epithelial cells help to maintain low glucose concentrations in human ASL which is important for protecting the lung against infection.

Whole-body heat stress and exercise stimulate the appearance of platelet microvesicles in plasma with limited influence of vascular shear stress.

Intense, large muscle mass exercise increases circulating microvesicles, but our understanding of microvesicle dynamics and mechanisms inducing their release remains limited. However, increased vascular shear stress is generally thought to be involved. Here, we manipulated exercise-independent and exercise-dependent shear stress using systemic heat stress with localized single-leg cooling (low shear) followed by single-leg knee extensor exercise with the cooled or heated leg (Study 1, n = 8) and whole-body passive heat stress followed by cycling (Study 2, n = 8). We quantified femoral artery shear rates (SRs) and arterial and venous platelet microvesicles (PMV-CD41+) and endothelial microvesicles (EMV-CD62E+). In Study 1, mild passive heat stress while one leg remained cooled did not affect [microvesicle] (P ≥ 0.05). Single-leg knee extensor exercise increased active leg SRs by ~12-fold and increased arterial and venous [PMVs] by two- to threefold, even in the nonexercising contralateral leg (P < 0.05). In Study 2, moderate whole-body passive heat stress increased arterial [PMV] compared with baseline (mean±SE, from 19.9 ± 1.5 to 35.5 ± 5.4 PMV.µL-1.103, P < 0.05), and cycling with heat stress increased [PMV] further in the venous circulation (from 27.5 ± 2.2 at baseline to 57.5 ± 7.2 PMV.µL-1.103 during cycling with heat stress, P < 0.05), with a tendency for increased appearance of PMV across exercising limbs. Taken together, these findings demonstrate that whole-body heat stress may increase arterial [PMV], and intense exercise engaging either large or small muscle mass promote PMV formation locally and systemically, with no influence upon [EMV]. Local shear stress, however, does not appear to be the major stimulus modulating PMV formation in healthy humans.

Whole body hyperthermia, but not skin hyperthermia, accelerates brain and locomotor limb circulatory strain and impairs exercise capacity in humans.

Cardiovascular strain and hyperthermia are thought to be important factors limiting exercise capacity in heat-stressed humans, however, the contribution of elevations in skin (Tsk) versus whole body temperatures on exercise capacity has not been characterized. To ascertain their relationships with exercise capacity, blood temperature (TB), oxygen uptake (V̇O2), brain perfusion (MCA Vmean), locomotor limb hemodynamics, and hematological parameters were assessed during incremental cycling exercise with elevated skin (mild hyperthermia; HYPmild), combined core and skin temperatures (moderate hyperthermia; HYPmod), and under control conditions. Both hyperthermic conditions increased Tsk versus control (6.2 ± 0.2°C; P < 0.001), however, only HYPmod increased resting TB, leg blood flow and cardiac output (Q̇), but not MCA Vmean Throughout exercise, Tsk remained elevated in both hyperthermic conditions, whereas only TB was greater in HYPmod At exhaustion, oxygen uptake and exercise capacity were reduced in HYPmod in association with lower leg blood flow, MCA Vmean and mean arterial pressure (MAP), but similar maximal heart rate and TB The attenuated brain and leg perfusion with hyperthermia was associated with a plateau in MCA and two-legged vascular conductance (VC). Mechanistically, the falling MCA VC was coupled to reductions in PaCO2, whereas the plateau in leg vascular conductance was related to markedly elevated plasma [NA] and a plateau in plasma ATP These findings reveal that whole-body hyperthermia, but not skin hyperthermia, compromises exercise capacity in heat-stressed humans through the early attenuation of brain and active muscle blood flow.

Loss of ecto-5'nucleotidase from porcine endothelial cells after exposure to human blood: Implications for xenotransplantation.

The endothelial cell surface expression of ecto-5'-nucleotidase (E5'N, CD73) is thought to be essential for the extracellular formation of cytoprotective, anti-thrombotic and immunosuppressive adenosine. Decreased E5'N activity may play a role in xenograft acute vascular rejection, preventing accommodation and tolerance mechanisms. We investigated the extent of changes in E5'N activity and other enzymes of purine metabolism in porcine hearts or endothelial cells when exposed to human blood or plasma and studied the role of humoral immunity in this context. Pig hearts, wild type (WT, n = 6) and transgenic (T, n = 5) for human decay accelerating factor (hDAF), were perfused ex vivo with fresh human blood for 4 h. Pig aortic endothelial cells (PAEC) were exposed for 3 h to autologous porcine plasma (PP), normal (NHP) or heat inactivated human plasma (HHP), with and without C1-inhibitor. Enzyme activities were measured in heart or endothelial cell homogenates with an HPLC based procedure. The baseline activity of E5'N in WT and T porcine hearts were 6.60 +/- 0.33 nmol/min/mg protein and 8.54 +/- 2.10 nmol/min/mg protein respectively (P < 0.01). Ex vivo perfusion of pig hearts with fresh human blood for 4 h resulted in a decrease in E5'N activity to 4.01 +/- 0.32 and 4.52 +/- 0.52 nmol/min/mg protein (P < 0.001) in WT and T hearts respectively, despite attenuation of hyperacute rejection in transgenic pigs. The initial PAEC activity of E5'N was 9.10 +/- 1.40 nmol/min/mg protein. Activity decreased to 6.76 +/- 0.57 and 4.58 +/- 0.47 nmol/min/mg protein (P < 0.01) after 3 h exposure of HHP and NHP respectively (P < 0.05), whereas it remained unchanged at 9.62 +/- 0.88 nmol/min/mg protein when incubated with PP controls. C1-inhibitor partially preserved E5'N activity, similar to the effect of HHP. Adenosine deaminase, adenosine kinase and AMP deaminase (other enzymes of purine metabolism) showed a downward trend in activity, but none were statistically significant. We demonstrate a specific decrease in E5'N activity in pig hearts following exposure to human blood which impairs adenosine production resulting in a loss of a cytoprotective phenotype, contributing to xenograft rejection. This effect is triggered by human humoral immune responses, and complement contributes but does not fully mediate E5'N depletion.

Hyperglycaemia and Pseudomonas aeruginosa acidify cystic fibrosis airway surface liquid by elevating epithelial monocarboxylate transporter 2 dependent lactate-H+ secretion.

The cystic fibrosis (CF) airway surface liquid (ASL) provides a nutrient rich environment for bacterial growth including elevated glucose, which together with defective bacterial killing due to aberrant HCO3- transport and acidic ASL, make the CF airways susceptible to colonisation by respiratory pathogens such as Pseudomonas aeruginosa. Approximately half of adults with CF have CF related diabetes (CFRD) and this is associated with increased respiratory decline. CF ASL contains elevated lactate concentrations and hyperglycaemia can also increase ASL lactate. We show that primary human bronchial epithelial (HBE) cells secrete lactate into ASL, which is elevated in hyperglycaemia. This leads to ASL acidification in CFHBE, which could only be mimicked in non-CF HBE following HCO3- removal. Hyperglycaemia-induced changes in ASL lactate and pH were exacerbated by the presence of P. aeruginosa and were attenuated by inhibition of monocarboxylate lactate-H+ co-transporters (MCTs) with AR-C155858. We conclude that hyperglycaemia and P. aeruginosa induce a metabolic shift which increases lactate generation and efflux into ASL via epithelial MCT2 transporters. Normal airways compensate for MCT-driven H+ secretion by secreting HCO3-, a process which is dysfunctional in CF airway epithelium leading to ASL acidification and that these processes may contribute to worsening respiratory disease in CFRD.

Decreased cardiac activity of AMP deaminase in subjects with the AMPD1 mutation--a potential mechanism of protection in heart failure.

OBJECTIVES: Possession of the C34T (Glu12Stop) nonsense mutation in the AMP-deaminase 1 (AMPD1) gene has been shown to be associated with improved prognosis in heart failure and ischemic heart disease. The most likely event leading to these clinical effects is a reduced capacity of the AMP deamination pathway and increased production of cardio-protective adenosine. However, since AMPD1 is predominantly expressed in skeletal muscle, the protective effects could be related not only to local cardiac changes, but also to a systemic mechanism. In the present study we evaluated the effect of the C34T mutation on cardiac AMP-deaminase activity and on the systemic changes in adenosine production. METHODS: The presence of the C34T mutation was assayed by single-stranded conformational polymorphism (SSCP). Analysis of the AMPD1 genotype and measurement of enzyme activities was performed on 27 patients with heart failure (HF). In addition, blood adenosine concentration was measured by liquid chromatography/mass spectrometry (LC/MS) in 21 healthy subjects with established AMPD1 genotype at rest and following exhaustive exercise. RESULTS: Cardiac AMP-deaminase activity in heterozygotes (C/T) was 0.59+/-0.02 nmol/min/g wet wt-about half of the activity found in normal wild-type (C/C) individuals (1.06+/-0.09 nmol/min/g wet wt, P=0.003). There were no significant differences in the activities of any other enzymes between subjects with the C/T or C/C genotype. Resting venous blood adenosine concentration was similar in subjects with C/C, C/T and homozygous for the mutated allele (T/T) genotype. Following exercise, a significant increase in adenosine was observed in T/T subjects (by 0.013+/-0.009 micromol/l, P=0.035) but not in C/C (0.003+/-0.009 micromol/l) or C/T (-0.002+/-0.011 micromol/l). CONCLUSIONS: Our findings indicate that the C34T mutation of AMPD1 leads to a decrease in cardiac enzyme activity of AMP-deaminase without changes in any other adenosine-regulating enzymes, highlighting the importance of local cardiac metabolic changes. Systemic (blood) changes in adenosine concentration were apparent only in homozygous subjects and therefore may play a relatively small part in cardio-protection.

C34T AMP deaminase 1 gene mutation protects cardiac function in donors.

Dysfunction of the donor heart is an important clinical problem that could be affected by genetic factors. We tested the hypothesis that possession of the C34T nonsense mutation in AMPD1 gene, which is known to improve survival in chronic heart failure, protects against cardiac dysfunction in donors. Genetic analysis for C34T mutation was performed by single-stranded conformational polymorphism (SSCP) in 22 donor hearts used for transplantation, 10 unused donor hearts with acute heart failure (HF), 37 patients with chronic HF, and 207 healthy controls. We found a significantly higher frequency of the mutation among donors with healthy hearts used for transplantation (31.8%) as compared to control population (13.5%, P < 0.001) and a lower frequency in dysfunctional donor hearts (5.0% P = 0.025); the frequency of the C34T mutation in patients with chronic heart failure (14.8%) was not different from that of a control population. The presence of the C34T mutation in AMPD1 gene appears to be protective against acute heart failure in cardiac donors.

Apical and basolateral localisation of GLUT2 transporters in human lung epithelial cells.

Glucose concentrations of normal human airway surface liquid are approximately 12.5 times lower than blood glucose concentrations indicating that glucose uptake by epithelial cells may play a role in maintaining lung glucose homeostasis. We have therefore investigated potential glucose uptake mechanisms in non-polarised and polarised H441 human airway epithelial cells and bronchial biopsies. We detected mRNA and protein for glucose transporter type 2 (GLUT2) and glucose transporter type 4 (GLUT4) in non-polarised cells but GLUT4 was not detected in the plasma membrane. In polarised cells, GLUT2 protein was detected in both apical and basolateral membranes. Furthermore, GLUT2 protein was localised to epithelial cells of human bronchial mucosa biopsies. In non-polarised H441 cells, uptake of D: -glucose and deoxyglucose was similar. Uptake of both was inhibited by phloretin indicating that glucose uptake was via GLUT-mediated transport. Phloretin-sensitive transport remained the predominant route for glucose uptake across apical and basolateral membranes of polarised cells and was maximal at 5-10 mM glucose. We could not conclusively demonstrate sodium/glucose transporter-mediated transport in non-polarised or polarised cells. Our study provides the first evidence that glucose transport in human airway epithelial cells in vitro and in vivo utilises GLUT2 transporters. We speculate that these transporters could contribute to glucose uptake/homeostasis in the human airway.

Mammalian mismatches in nucleotide metabolism: implications for xenotransplantation.

Acute humoral rejection (AHR) limits the clinical application of animal organs for xenotransplantation. Mammalian disparities in nucleotide metabolism may contribute significantly to the microvascular component in AHR; these, however remain ill-defined. We evaluated the extent of species-specific differences in nucleotide metabolism. HPLC analysis was performed on venous blood samples (nucleotide metabolites) and heart biopsies (purine enzymes) from wild type mice, rats, pigs, baboons, and human donors.Ecto-5'-nucleotidase (E5'N) activities were 4-fold lower in pigs and baboon hearts compared to human and mice hearts while rat activity was highest. Similar differences between pigs and humans were also observed with kidneys and endothelial cells. More than 10-fold differences were observed with other purine enzymes. AMP deaminase (AMPD) activity was exceptionally high in mice but very low in pig and baboon hearts. Adenosine deaminase (ADA) activity was highest in baboons. Adenosine kinase (AK) activity was more consistent across different species. Pig blood had the highest levels of hypoxanthine, inosine and adenine. Human blood uric acid concentration was almost 100 times higher than in other species studied. We conclude that species-specific differences in nucleotide metabolism may affect compatibility of pig organs within a human metabolic environment. Furthermore, nucleotide metabolic mismatches may affect clinical relevance of animal organ transplant models. Supplementation of deficient precursors or application of inhibitors of nucleotide metabolism (e.g., allopurinol) or transgenic upregulation of E5'N may overcome some of these differences.