Intracellular Concentration Of Adenosine Research Articles

Metabolic stress-induced human beta-cell death is mediated by increased intracellular levels of adenosine.

High intracellular concentrations of adenosine and 2'-deoxyadenosine have been suggested to be an important mediator of cell death. The aim of the present study was to characterize adenosine-induced death in insulin-producing beta-cells, at control and high glucose + palmitate-induced stress conditions. Human insulin-producing EndoC-betaH1 cells were treated with adenosine, 2'-deoxyadenosine, inosine and high glucose + sodium palmitate, and death rates using flow cytometry were studied. We observed that adenosine and the non-receptor-activating analogue 2-deoxyadenosine, but not the adenosine deamination product inosine, promoted beta-cell apoptosis at concentrations exceeding maximal adenosine-receptor stimulating concentrations. Both adenosine and inosine were efficiently taken up by EndoC-betaH1 cells, and inosine counteracted the cell death promoting effect of adenosine by competing with adenosine for uptake. Both adenosine and 2'-deoxyadenosine promptly reduced insulin-stimulated production of plasma membrane PI(3,4,5)P3, an effect that was reversed upon wash out of adenosine. In line with this, adenosine, but not inosine, rapidly diminished Akt phosphorylation. Both pharmacological Bax inhibition and Akt activation blocked adenosine-induced beta-cell apoptosis, indicating that adenosine/2'-deoxyadenosine inhibits the PI3K/Akt/BAD anti-apoptotic pathway. High glucose + palmitate-induced cell death was paralleled by increased intracellular adenosine and inosine levels. Overexpression of adenosine deaminase-1 (ADA1) in EndoC-betaH1 cells, which increased Akt phosphorylation, prevented both adenosine-induced apoptosis and high glucose + palmitate-induced necrosis. ADA2 overexpression not only failed to protect against adenosine and high glucose + palmitate-activated cell death, but instead potentiated the apoptosis-stimulating effect of adenosine. In line with this, ADA1 overexpression increased inosine production from adenosine-exposed cells, whereas ADA2 did not. Knockdown of ADA1 resulted in increased cell death rates in response to both adenosine and high glucose + palmitate. Inhibition of miR-30e-3p binding to the ADA1 mRNA 3'-UTR promoted the opposite effects on cell death rates and reduced intracellular adenosine contents. It is concluded that intracellular adenosine/2'-deoxyadenosine regulates negatively the PI3K pathway and is therefore an important mediator of beta-cell apoptosis. Adenosine levels are controlled, at least in part, by ADA1, and strategies to upregulate ADA1 activity, during conditions of metabolic stress, could be useful in attempts to preserve beta-cell mass in diabetes.

Article Review: Autosomal Dominant Polycystic Kidney Disease: Renal Physiology Diagnosis, Treatment and Novel Therapies

In the case of Autosomal dominant polycystic kidney disease, patients are usually cured with Vasopressin (V2) receptor antagonists, which delay the ongoing growth of cyst formation and slow the pace of AD disease progression. Before we know more, it is uncertain if the increase in vasopressin amide levels that was detected during V2RAT treatment impacts the production of glucose in the intestines. Cell growth and fluid secretion are aided by high intracellular concentrations of adenosine 3',5;-cyclic monophosphate (cAMP), which leads to cyst development. SST, a hormone implicated in a variety of cell activities, has the potential to block the generation of intracellular cAMP. Nevertheless, since Somatostatin is quickly removed in vivo, it has little therapeutic promise. As a result, analogues with a longer half-life have been established, which might be potential medicines in the therapy of ADPKD. This review covers the complicated physiological consequences of Somatostatin, especially on the kidneys, as well as the possible therapeutic use of SST analogues in ADPKD.

Adenosine-regulated mechanisms in the pathogenesis of ventilation disorders in patients with pulmonary tuberculosis

Uncovering involvement of the purinergic system in the pathogenesis of ventilation disorders (VD) may provide additional information about the pathophysiological mechanisms leading to the development of VD in pulmonary tuberculosis (PT). The aim was to identify a relationship between the parameters of adenosine metabolism, inflammatory response and altered ventilation metabolism in PT patients. Materials and methods. Obstructive and mixed PT patients were assigned to subgroups with/without VD for assessing adenosine deaminase activity (ADA-1, 2) in serum, mononuclear cells, neutrophils; ecto-5’-nucleotidase (ecto-5’-NT); CD26 (dipeptidyl peptidase-4, DPP-4), phagocyte oxidative burst measured by NO generation. Results. PT patients showed decreased ADA-1 and CD26 (DPP-4), but increased ADA-2. Elevated intracellular adenosine concentration was found in mononuclear cells in patients lacking VD, whereas patients with mixed and obstructive VD — had it in neutrophils. Mononuclear cells of patients with PT lacking VD as well as with obstructive VD type had decreased NO3– concentration. Neutrophil hyperactivity was recorded in all groups of PT patients. Patients with PT lacking VD as well as with mixed VD type showed that the parameters of external respiration were associated with activity of extra-/intracellular ADA, whereas obstructive VD was caused by excessive formation of serum adenosine. Changes in respiratory function in PT were associated with decreased level of serum NO radicals, impaired nitrogen-dependent bactericidal phagocyte activity, and overproduced neutrophil oxygen radicals. Conclusion. Purinergic regulation is involved in regulating inflammatory and compensatory processes in PT patients as well as impaired ventilation efficiency. The most severe respiratory disorders observed in PT patients with mixed VD type are associated with the most prominent changes in nucleotidase activity, particularly ecto-ADA-2 and DPP-4/CD26.

Intracellular ATP levels in mouse cortical excitatory neurons varies with sleep\u2013wake states

Whilst the brain is assumed to exert homeostatic functions to keep the cellular energy status constant under physiological conditions, this has not been experimentally proven. Here, we conducted in vivo optical recordings of intracellular concentration of adenosine 5’-triphosphate (ATP), the major cellular energy metabolite, using a genetically encoded sensor in the mouse brain. We demonstrate that intracellular ATP levels in cortical excitatory neurons fluctuate in a cortex-wide manner depending on the sleep-wake states, correlating with arousal. Interestingly, ATP levels profoundly decreased during rapid eye movement sleep, suggesting a negative energy balance in neurons despite a simultaneous increase in cerebral hemodynamics for energy supply. The reduction in intracellular ATP was also observed in response to local electrical stimulation for neuronal activation, whereas the hemodynamics were simultaneously enhanced. These observations indicate that cerebral energy metabolism may not always meet neuronal energy demands, consequently resulting in physiological fluctuations of intracellular ATP levels in neurons.

The Effect of Equilibriative Nucleoside Transporter 1 on Pseudomonas Aeruginosa‐Induced Acute Lung Injury

Acute lung injury (ALI) is hallmarked by lung edema, excessive inflammation, and respiratory failure resulting in 40% mortality. Multidrug resistant Pseudomonas aeruginosa (P. aeruginosa) infections often cause ALI in patients with compromised immunity. It is well established that Adenosine, a signaling nucleoside increased upon tissue injury, has been shown to protect against ALI through activation of adenosine receptors (ARs). Extracellular and intracellular adenosine concentrations are regulated by equilibriative nucleoside transporters (ENTs). Whether altering adenosine signaling and uptake can prevent or treat bacterial P. aeruginosa‐induced ALI is unknown. We hypothesize that ENT1 inhibition will increase extracellular adenosine and protect against P. aeruginosa‐induced ALI via activation of ARs. ENT1 pharmacological inhibitors and null mice were used to determine the effects of ENT1 inhibition on P. aeruginosa‐induced ALI. We found that P. aeruginosa infections induced ALI in C57BL/6 mice in a dose dependent manner. Pharmacological inhibition of ENT1 via NBTI significantly attenuated P. aeruginosa‐induced ALI, as assessed by wet‐to‐dry lung weight, bronchoalveolar lavage (BAL) protein levels, BAL inflammatory cells, and pulmonary function determined by static lung compliance, tissue damping, and tissue elastance. NBTI also increased bacterial clearance in mice infected with P. aeruginosa. Additionally, NBTI attenuated P. aeruginosa‐induced IL‐1β levels in the BAL of mice. Consistent with ENT1 pharmacological inhibitor data, ENT1 null mice attenuated P. aeruginosa‐induced ALI as assessed by BAL protein and BAL cell counts. Finally, agonists of A2AR and A2BR mimicked NBTI blunting of P. aeruginosa‐induced ALI, whereas, antagonists against A2AR or A2BR diminished the protective effect of NBTI inhibition on P. aeruginosa‐induced ALI. These results suggest that inhibition of ENT1 protects against P. aeruginosa‐induced ALI via adenosine‐mediated activation of A2AR and A2BR. Inhibition of ENT1 may be a novel approach to prevent and treat P. aeruginosa‐ induced ALI.Support or Funding InformationThis work was in part supported by NIGMS P20GM103652 to S. Rounds and Q. Lu, RO1 HL130230 to Q. Lu, PULM‐019‐14F to S. Rounds, and NIEHS T32 ES007272 to E. Chambers.

An Adenosine-Mediated Glial-Neuronal Circuit for Homeostatic Sleep.

The work presented here provides evidence for an adenosine-mediated regulation of sleep in response to waking (i.e., homeostatic sleep need), requiring activation of neuronal adenosine A1 receptors and controlled by glial adenosine kinase. Adenosine kinase acts as a highly sensitive and important metabolic sensor of the glial ATP/ADP and AMP ratio directly controlling intracellular adenosine concentration. Glial equilibrative adenosine transporters reflect the intracellular concentration to the extracellular milieu to activate neuronal adenosine receptors. Thus, adenosine mediates a glial-neuronal circuit linking glial metabolic state to neural-expressed sleep homeostasis. This indicates a metabolically related function(s) for this glial-neuronal circuit in the buildup and resolution of our need to sleep and suggests potential therapeutic targets more directly related to sleep function.

Adenosine Deaminase Inhibitor EHNA Exhibits a Potent Anticancer Effect Against Malignant Pleural Mesothelioma

Background/Aims: Malignant pleural mesothelioma (MPM) is an aggressive malignant tumor and an effective therapy has been little provided as yet. The present study investigated the possibility for the adenosine deaminase (ADA) inhibitor EHNA as a target of MPM treatment. Methods: MTT assay, TUNEL staining, monitoring of intracellular adenosine concentrations, and Western blotting were carried out in cultured human MPM cell lines without and with knocking-down ADA. The in vivo effect of EHNA was assessed in mice inoculated with NCI-H2052 MPM cells. Results: EHNA induced apoptosis of human MPM cell lines in a concentration (0.01-1 mM)- and treatment time (24-48 h)-dependent manner, but such effect was not obtained with another ADA inhibitor pentostatin. EHNA increased intracellular adenosine concentrations in a treatment time (3-9 h)-dependent manner. EHNA-induced apoptosis of MPM cells was mimicked by knocking-down ADA, and the effect was neutralized by the adenosine kinase inhibitor ABT-702. EHNA clearly suppressed tumor growth in mice inoculated with NCI-H2052 MPM cells. Conclusion: The results of the present study show that EHNA induces apoptosis of MPM cells by increasing intracellular adenosine concentrations, to convert to AMP, and effectively prevents MPM cell proliferation. This suggests that EHNA may be useful for treatment of the tragic neoplasm MPM.

The effects of N-acylhomoserine lactones, β-lactam antibiotics and adenosine on biofilm formation in the multi-β-lactam antibiotic-resistant bacterium Acidovorax sp. strain MR-S7

Bacteria in the natural ecosystem frequently live as adherent communities called biofilms. Some chemical compounds are known to affect biofilm formation. We investigated the effect of exogenous small molecules, N-acylhomoserine lactones (AHLs), β-lactam antibiotics, and adenosine, on biofilm formation in the β-lactam antibiotic-resistant bacterium Acidovorax sp. strain MR-S7. Biofilm formation was induced by the addition of various types of AHL isomers and β-lactam antibiotics, whereas the addition of adenosine strongly interfered with the biofilm formation. A gene (macP) encoding adenosine deaminase (that converts adenosine to inosine controlling intracellular adenosine concentration) was successfully cloned from MR-S7 genome and heterologously expressed in Escherichia coli. The purified MacP protein clearly catalyzed the deamination of adenosine to produce inosine. A transcriptional analysis revealed that biofilm-inducing molecules, an AHL and a β-lactam antibiotic, strongly induced not only biofilm formation but also adenosine deaminase gene expression, suggesting that an elaborate gene regulation network for biofilm formation is present in the β-lactam antibiotic-resistant bacterium studied here.

Reactivation of the Tumour Suppressor RASSF1A in Breast Cancer by Simultaneous Targeting of DNA and E2F1 Methylation

BackgroundTumour suppressor genes are often transcriptionally silenced by promoter hypermethylation, and recent research has implicated alterations in chromatin structure as the mechanistic basis for this repression. In addition to DNA methylation, other epigenetic post-translational modifications that modulate the stability and binding of specific transcription factors to gene promoters have emerged as important mechanisms for controlling gene expression. The aim of this study was to analyse the implications of these mechanisms and their molecular connections in the reactivation of RASSF1A in breast cancer.MethodsCompounds that modulate the intracellular concentration of adenosine, such as dipyridamole (DIPY), greatly increase the antiproliferative effects of 3-O-(3,4,5-trimethoxybenzoyl)-(−)-catechin (TMCG), a synthetic antifolate derived from the structure of tea catechins. Quantitative real-time PCR arrays and MALDI-TOF mass spectrometry indicated that this combination (TMCG/DIPY) induced apoptosis in breast cancer cells by modulating the methylation levels of DNA and proteins (such as E2F1), respectively. Chromatin immunoprecipitation (ChIP) assays were employed to confirm that this combination induced chromatin remodelling of the RASSF1A promoter and increased the occupancy of E2F1 at the promoter of this tumour suppressor gene.ResultsThe TMCG/DIPY combination acted as an epigenetic treatment that reactivated RASSF1A expression and induced apoptosis in breast cancer cells. In addition to modulating DNA methylation and chromatin remodelling, this combination also induced demethylation of the E2F1 transcription factor. The ChIP assay showed enhancement of E2F1 occupancy at the unmethylated RASSF1A promoter after TMCG/DIPY treatment. Interestingly, inhibition of E2F1 demethylation using an irreversible inhibitor of lysine-specific demethylase 1 reduced both TMCG/DIPY-mediated RASSF1A expression and apoptosis in MDA-MB-231 cells, suggesting that DNA and protein demethylation may act together to control these molecular and cellular processes.Conclusions/SignificanceThis study demonstrates that simultaneous targeting of DNA and E2F1 methylation is an effective epigenetic treatment that reactivates RASSF1A expression and induces apoptosis in breast cancer cells.

Caspase-4 Activation in Association with Decreased Adenosine Deaminase Activity May Be a Factor for Gastric Ulcer

Background/Aims: Adenosine deaminase (ADA)-deficient disease, a severe combined immunodeficiency, is most commonly associated with gastrointestinal disorders such as ulcer. The present study investigated the role of ADA in the pathogenesis of gastric ulcer. Methods: ADA activity was measured in a variety of organs and tissues from rats and in human gastric biopsy samples from patients who underwent gastrofiberscope. An MTT assay and TUNEL staining were carried out, and activity of caspase-3 and -4 was enzymatically measured in MKN45 human gastric cancer cells. In the Western blot analysis, caspase-4 activation was identified in gastric biopsy samples. Results: In rat organs and tissues, the epithelium of the gastrointestine exhibited higher ADA activity. The ADA inhibitor EHNA reduced cell viability, increased TUNEL-positive cells, and activated caspase-3 and -4 in MKN45 cells. For gastric biopsy samples, much lower ADA activity was found in gastric ulcer tissues, with a tendency of caspase-4 activation. Conclusion: A decline in ADA activity and the ensuing increase in intracellular adenosine concentrations for the stomach could induce gastric epithelial cell apoptosis by activating caspase-4 and the effector caspase-3. This may represent a fresh pathogenetical pathway for gastric ulcer relevant to ADA activity and caspase-4 activation.

Nucleoside Transporter Proteins

Concentrative nucleoside transporters (CNT; SLC28) and equilibrative nucleoside transporters (ENT; SLC29) mediate the uptake of natural nucleosides and a variety of nucleoside-derived drugs, mostly used in anticancer therapy. SLC28 and SLC29 families consist in three and four members, respectively, which differ in their substrate selectivity and their energy requirements. Tissue distribution of these transporters is not homogeneous among tissues, and their expression can be regulated. In epithelia, CNT and ENT proteins are mostly localized in the apical and basolateral membranes, respectively, which results in nucleoside and nucleoside-derived drugs vectorial flux. Nucleoside transporters can play physiological roles other than salvages, such as the modulation of extracellular and intracellular adenosine concentrations. Moreover, these transporters also have clinical significance. ENT proteins are target of dipyridamole and dilazep, used as vasodilatory drugs in the treatment of heart and vascular diseases. On the other hand, nucleoside transporters are responsible for the cellular uptake of currently used anticancer nucleoside-derived drugs, thus these membrane proteins might play a significant role in nucleoside-based chemotherapy. Finally, several polymorphisms have been described in CNT and ENT proteins that could affect nucleoside homeostasis, adenosine signalling events or nucleoside-derived drug cytotoxicity or pharmacokinetics.

Targeting the methionine cycle for melanoma therapy with 3‐O‐(3,4,5‐trimethoxybenzoyl)‐(−)‐epicatechin

The higher expression of methionine cycle genes in melanoma cells than in normal melanocytes may be related with increased protein synthesis and transmethylation reactions and the subsequent need for high levels of methionine. 3-O-(3,4,5-trimethoxybenzoyl)-(-)-epicatechin (TMECG), a trimethoxy derivative of epicatechin-3-gallate (ECG), effectively suppressed proliferation of melanoma cells in cultures by inducing apoptosis. TMECG modulates the expression of genes involved in methionine metabolism, cellular methylation and glutathione synthesis in melanoma cells. TMECG treatment of melanoma cells resulted in the downregulation of antiapoptotic Bcl-2, the upregulation of proapoptotic Bax and the activation of caspase-3; however, it did not induce the expression of the apoptosis protease-activating factor-1 (Apaf-1). Having elucidated the effects of TMECG on the melanoma methionine cycle, we designed therapeuthical strategies to increase its effectiveness. Combinations of TMECG with S-adenosylmethionine or compounds that modulate the intracellular concentration of adenosine strongly increase the antiproliferative effects of TMECG. The ability of TMECG to target multiple aspects related with melanoma survival, with a high degree of potency, points to its clinical value in melanoma therapy.

Methotrexate induced differentiation in colon cancer cells is primarily due to purine deprivation

The folate antagonist methotrexate (MTX) inhibits synthesis of tetrahydrofolate (THF), pyrimidines and purines, and induces differentiation in several cell types. At 1 microM, MTX reduced proliferation and induced differentiation in HT29 colon cancer cells; the latter effect was augmented (P < 0.001) by thymidine (100 microM) but was reversed (P < 0.001) by the purines, hypoxanthine (Hx; 100 microM) and adenosine (100 microM). In contrast 5-fluoro-uracil (5-FU), a specific thymidylate synthase (TS) inhibitor, had no effect on differentiation, suggesting that MTX-induced differentiation is not due to a reduction in thymidine but to the inhibition of purine biosynthesis. Inhibition of cyclic AMP (cAMP) by RpcAMP (25 microM) further enhanced (P < 0.001) MTX induced differentiation, whereas the cAMP activator forskolin (10 microM) reversed (P < 0.001) MTX induced differentiation. These observations implicate a central role of adenosine and cAMP in MTX induced differentiation. By combining Western blot analysis with liquid chromatography-mass spectrometry (LC-MS)and HPLC analyses we also reveal both the expression and activity of key enzymes (i.e. methionine synthase (MS), s-adenosylhomocysteinase, cystathionine beta-synthase and ornithine decarboxylase) regulating methyl cycle, transsulfuration and polyamine pathways in HT29 colon cancer cells. At 1 microM, MTX induced differentiation was associated with a marked reduction in the intracellular concentrations of adenosine and, consequently, S-adenosylmethionine (SAM), S-adenosylhomocysteine, polyamines and glutathione (GSH). Importantly, the marked reduction in methionine that accompanied MS inhibition following MTX treatment was non-limiting with respect to SAM synthesis. Collectively, these findings indicate that the effects of MTX on cellular differentiation and single carbon metabolism are primarily due to the intracellular depletion of purines.

Increased homocysteine and decreased adenosine formation in Alzheimer's disease

Vascular risk factors increase the risk of Alzheimer's disease. Increased concentrations of circulating homocysteine are associated with vascular risk factors and Alzheimer's disease but the underlying mechanisms are unclear. Homocysteine inhibits the hydrolysis of S-adenosylhomocysteine leading to a decrease in the intracellular adenosine concentration. Adenosine is an endogenous protective molecule against atherosclerotic and vaso-occlusive disorders that contribute to the pathology of Alzheimer's disease. In this study the concentrations of homocysteine and adenosine were determined in the plasma of 25 patients with Alzheimer's disease and 25 control subjects. There was a significant increase in the plasma concentration of homocysteine (p < 0.0001) and a significant decrease in the plasma concentration of adenosine (p < 0.001). In the combined Alzheimer and control groups a significant negative correlation was found between the plasma concentrations of homocysteine and adenosine (r = -0.769, p < 0.0001). There was also a significant negative correlation between the plasma concentrations of homocysteine and adenosine in the Alzheimer group (r =-0.773, p < 0.0001). The decrease in adenosine formation may play a role in the vascular pathology of homocysteine in Alzheimer's disease.

Leptin, and Lipid Oxidation Through AMPK

One of the serious effects of diabetes is the excess storage of fat not only in adipocytes, but also in other tissues (termed lipotoxicity). Type II diabetes patients are insensitive to treatment with insulin, and it is hypothesized that insulin resistance results from an excessive accumulation of lipids in nonadipocytes such that the lipids interfere with the action of insulin on those cells. The hormone leptin, which can regulate food intake and neuroendocrine activity, now appears to regulate lipid oxidation in the mitochondria of nonadipocytes. Minokoshi et al. found that leptin injection in mouse muscle in vivo resulted in increased intracellular concentrations of adenosine 5′-monophosphate (AMP), and activated the α2 adenosine 5′-monophosphate-activated kinase (AMPK)--a protein that regulates cellular energy metabolism--in mouse muscle. However, not only did leptin activate α2 AMPK immediately in muscle, it also indirectly caused long-term activation of AMPK (for several hours) as mediated through the hypothalamus. The hypothalamus-dependent activation of AMPK was mediated by the α-adrenergic pathway. Leptin-mediated activation of AMPK led to the phosphorylation (and inactivation) of acetyl coenzyme A carboxylase (ACC), an enzyme that inhibits the import of lipids into and the oxidation of lipids in the mitochondria, and led to increased lipid oxidation in vivo. Leptin failed to increase phosphorylation of ACC in cells that overexpressed a dominant-negative form of AMPK, suggesting that leptin acts through AMPK to inhibit ACC and increase lipid oxidation. Thus, designing therapeutic drugs that target the leptin-AMPK pathway may prove useful for some diabetic patients. Y. Minokoshi, Y.-B. Kim, O. D. Peroni, L. G. D. Fryer, C. Müller, D. Carling, B. B. Kahn, Leptin stimulates fatty-acid oxidation by activating AMP-activated protein kinase. Nature 415 , 339-343 (2002). [Online Journal] J. Friedman, Fat in all the wrong places. Nature 415 , 268-269 (2002). [Online Journal]

An Anion Channel in Guinea Pig Gallbladder Epithelial Cells Is Highly Permeable to HCO−3

In guinea pig gallbladder epithelium, a secretion of fluid, secondary to an electrogenic secretion of Cl− and HCO−3, is elicited in the presence of a high intracellular concentration of adenosine 3′-5′-cyclic monophosphate (cAMP). The aim of this study was to analyze the effects of secretagogues on the activity of anionic channels in isolated epithelial cells using the patch-clamp technique and measuring the electrical potential difference of the cellular membrane (pdcm). In cell-attached configuration, with the microelectrode filled with a solution of N-methylglucamine–Cl, or in inside-out configuration (symmetrical solution), it was possible to demonstrate the presence of an 18-pS Cl− channel with linear current/voltage (I/V) relationship and voltage independence; this channel is not activated by cAMP (cell-attached configuration). In inside-out configuration (symmetrical solution), another anionic channel with a conductance of 2.8 pS, voltage independence, and a linear I/V relationship was also identified. This channel was stimulated by cAMP (cell-attached configuration) and by PKA + ATP + cAMP (inside-out configuration). The channel was inhibited by NPPB (10−5 M), but not by other anionic inhibitors. Measurements of the pdcm value suggested that in isolated cells, as in whole tissue, cAMP activates conductance for both Cl− and HCO−3. The selectivity of the channel was gluconate < SO2−4 < Cl− < Br− < I− < HCO−3 < SCN− and the PHCO3/PCl was 2.6. Some features of the channel resemble those of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel and RT-PCR performed on mRNA from isolated epithelial cells detected the presence of a CFTR homologue mRNA. The results obtained indicate that this channel is responsible for the HCO−3 conductance activated by cAMP.

Metabolic flux rates of adenosine in the heart.

The quantitatively most important source of adenosine under well-oxygenated conditions is 5'-AMP hydrolyzed by cytosolic 5'-nucleotidase N-I. Hydrolysis of S-adenosylhomocysteine and extracellular dephosphorylation of 5'-AMP further contribute to total production. More than 90% of the total production occur intracellularly under well-oxygenated conditions. Besides cardiomyocytes, endothelial cells and smooth muscle contribute significantly to total cardiac adenosine production. Rapid enzymatic conversion of adenosine is provided by adenosine kinase and adenosine deaminase, keeping the cytosolic adenosine concentration in the nanomolar range. Due to the high intracellular rates of adenosine rephosphorylation and deamination the cytosolic is normally below the extracellular adenosine concentration, making the cytosol to a sink rather than a source of adenosine. It is for this reason that blockers of membrane transport enhance the plasma adenosine concentration. With increasing catabolism of adenine nucleotides the rate of intracellular adenosine production exceeds the rate of adenosine deamination and rephosphorylation. Thus, this condition will result in a concentration gradient from intra- to extracellular. Thence, membrane transport blockers would be expected to increase the intracellular adenosine concentration. A considerable insecurity on the importance of experimental data results from species differences of purine metabolism. Cardiac adenosine metabolism has recently been described in quantitative terms using mathematical model analysis. This analysis tool may prove useful in future when (1) clarifying the importance of various regulatory actions described for the different pathways of adenosine metabolism, (2) making quantitative comparisons of different experimental models possible and (3) deepening the insight from experimental data.

Coronary reserve of high- and low-flow regions in the dog heart left ventricle.

Left ventricular myocardial blood flow is spatially heterogeneous. The hypothesis we tested was whether myocardial areas with a steady-state flow <0.5 times mean flow are underperfused and areas with flow > 1.5 times mean flow are overperfused. In anesthetized beagle dogs (n=10), the relationship between local blood flow versus S-adenosylhomocysteine (SAH) concentration, a measure of the free intracellular adenosine concentration, and lactate, a measure of the myocardial NADH/NAD+ ratio, were determined under control conditions and after coronary constriction. Control local myocardial blood flow was 0.99+/-0.46 mL x min(-1) x g(-1), with a coefficient of variation of 0.36+/-0.12 (n=256 per heart; sample wet mass, 125+/-30 mg). Tissue concentrations of SAH (3.4+/-2.5 nmol/g) and lactate (1.88+/-0.80 micromol/g) were not elevated in low-flow samples. However, after coronary artery constriction, poststenotic blood flow decreased from 1.00+/-0.27 to 0.49+/-0.22 mL x min(-1) x g(-1) (P<0.04), with significant correlation between local SAH and flow (r=-0.59) and lactate and flow (r = -0.50). Although nearly all samples from control high-flow regions showed increased SAH concentrations if relative flow after stenosis was <1.0, control low-flow samples frequently displayed low SAH concentrations. The percent reduction in flow determined the changes in the local SAH and lactate concentration, independent of the local control blood flow. When the coronary inflow is unrestricted, the oxygen supply to control low-flow regions meets metabolic demand. Flow to control high-flow regions reflects a higher local demand rather than overperfusion. Thus, blood flow heterogeneity most likely reflects differences in aerobic metabolism.

Survival of energy failure in the anoxic frog brain: delayed release of glutamate.

This study investigated the relationship between energy failure and neurotransmitter release in the frog (Rana pipiens) brain during 1-3 h of anoxia. Unlike truly anoxia-tolerant species, the frog does not defend its brain energy charge. When exposed to anoxia at 25 degrees C, there is an immediate fall in brain ATP levels, which reach approximately 20% of normoxic levels in approximately 60 min. The frog, nevertheless, survives another 1-2 h of anoxia. At 100 min of anoxia, there is an increase in extracellular adenosine concentration, probably originating from the increased intracellular adenosine concentration caused by the breakdown of intracellular ATP. Increases in the levels of extracellular glutamate and GABA do not occur until 1-2 h after ATP depletion. This response is quite unlike that recorded for other vertebrates, anoxia-tolerant or anoxia-intolerant, where energy failure quickly results in an uncontrolled and neurotoxic release of excitatory neurotransmitters. In the frog, the delay in excitotoxic neurotransmitter release may be one of the factors that allow a period of survival after energy failure. Clearly, energy failure by itself is not a fatal event in the frog brain.

The oligopeptide transporter (Pept-1) in human intestine: Biology and function

The oligopeptide transporter (Pept-1), which is located in the intestinal brush border membrane, provides a major mechanism for protein absorption in the human intestine. Expression cloning of the gene encoding Pept-1 has predicted a 78,810-kilodalton protein consisting of 708 amino acid residues and possessing 12 putative membrane-spanning domains. The characterization of its function by in vivo and in vitro studies has shown that (1) it transports dipeptides and tripeptides but not free amino acids or peptides with more than three amino acid residues, and (2) its driving force for uphill transport requires proton binding and presence of an inside-negative membrane potential. There has also been cloning of a membrane protein (HPT-1) which appears to be associated with the oligopeptide transporter. However, the nature of association has not yet been determined. A human intestinal cell line (Caco-2), which expresses Pept- 1, has been used to investigate the effects of metabolic and pathological factors on dipeptide transport. These studies suggest that the insulin stimulates dipeptide transport by increasing membrane insertion of oligopeptide transporter from a preformed cytoplasmic pool, and cholera toxin decreases dipeptide transport by inhibiting the activity of Pept-1 through an increase in the intracellular concentration of adenosine 3',5'-cyclic monophosphate. Lastly, Pept-1 seems to play important roles in nutritional and pharmacological therapies; for example, it has allowed the use of oligopeptides as a source of nitrogen for enteral feeding and the use of oral route for delivery of peptidomimetic drugs such as beta-lactam antibiotics. (Gastroenterology 1997 Jul;113(1):332-40)