Exposure to human blood inactivates swine endothelial ecto-5'-nucleotidase

Ecto-5'-nucleotidase (E5'N) is an extracellular enzyme forming anti-inflammatory and immunosuppressive adenosine. We evaluated whether confrontation of pig heart and endothelial cells with human blood changes the activity of E5'N. Pig hearts were perfused ex vivo with fresh human blood for 4 h. Pig aortic endothelial cells (PAEC) were incubated in vitro with human plasma for 3 h. Ex vivo perfusion of pig heart with fresh human blood resulted in a decrease in E5'N activity to 62% and 61% of initial in wild-type and transgenic pig hearts, respectively. PAEC activity of E5'N decreased to 71% and 50% of initial after 3 h exposure to heat-inactivated and active complement human plasma, respectively, while it remained constant in controls. Pig heart activity of E5'N decreased following exposure to human blood, which may affect adenosine production and exacerbate hyperacute and vascular rejection.     

Knockout of Kir6.2 negates ischemic preconditioning-induced protection of myocardial energetics

Although ischemic preconditioning induces bioenergetic tolerance and thereby remodels energy metabolism that is crucial for postischemic recovery of the heart, the molecular components associated with preservation of cellular energy production, transfer, and utilization are not fully understood. Here myocardial bioenergetic dynamics were assessed by (18)O-assisted (31)P-NMR spectroscopy in control or preconditioned hearts from wild-type (WT) or Kir6.2-knockout (Kir6.2-KO) mice that lack metabolism-sensing sarcolemmal ATP-sensitive K(+) (K(ATP)) channels. In WT vs. Kir6.2-KO hearts, preconditioning induced a significantly higher total ATP turnover (232 +/- 20 vs. 155 +/- 15 nmol x mg protein(-1) x min(-1)), ATP synthesis rate (58 +/- 3 vs. 46 +/- 3% (18)O labeling of gamma-ATP), and ATP consumption rate (51 +/- 4 vs. 31 +/- 4% (18)O labeling of P(i)) after ischemia-reperfusion. Moreover, preconditioning preserved cardiac creatine kinase-catalyzed phosphotransfer in WT (234 +/- 26 nmol x mg protein(-1) x min(-1)) but not Kir6.2-KO (133 +/- 18 nmol x mg protein(-1) x min(-1)) hearts. In contrast with WT hearts, preconditioning failed to preserve contractile recovery in Kir6.2-KO hearts, as tight coupling between postischemic performance and high-energy phosphoryl transfer was compromised in the K(ATP)-channel-deficient myocardium. Thus intact K(ATP) channels are integral in ischemic preconditioning-induced protection of cellular energetic dynamics and associated cardiac performance.     

Mechanism of decreased adenosine protection in reperfusion injury of aging rats

The purpose of this study was to determine whether the protective effects of adenosine on myocardial ischemia-reperfusion injury are altered with age, and if so, to clarify the mechanisms that underlie this change related to nitric oxide (NO) derived from the vascular endothelium. Isolated perfused rat hearts were exposed to 30 min of ischemia and 60 min of reperfusion. In the adult hearts, administration of adenosine (5 micromol/l) stimulated NO release (1. 06 +/- 0.19 nmol. min(-1). g(-1), P < 0.01 vs. vehicle), increased coronary flow, improved cardiac functional recovery (left ventricular developed pressure 79 +/- 3.8 vs. 57 +/- 3.1 mmHg in vehicle, P < 0.001; maximal rate of left ventricular pressure development 2,385 +/- 103 vs. 1,780 +/- 96 in vehicle, P < 0.001), and reduced myocardial creatine kinase loss (95 +/- 3.9 vs. 159 +/- 4.6 U/100 mg protein, P < 0.01). In aged hearts, adenosine-stimulated NO release was markedly reduced (+0.42 +/- 0.12 nmol. min(-1). g(-1) vs. vehicle), and the cardioprotective effects of adenosine were also attenuated. Inhibition of NO production in the adult hearts significantly decreased the cardioprotective effects of adenosine, whereas supplementation of NO in the aged hearts significantly enhanced the cardioprotective effects of adenosine. The results show that the protective effects of adenosine on myocardial ischemia-reperfusion injury are markedly diminished in aged animals, and that the loss in NO release in response to adenosine may be at least partially responsible for this age-related alteration.     

Absolute rates of adenosine formation during ischaemia in rat and pigeon hearts.

1. The activities of ecto- and cytosolic 5'-nucleotidase (EC 3.1.3.5), adenosine kinase (EC 2.7.1.20), adenosine deaminase (EC 3.5.4.4) and AMP deaminase (EC 3.5.4.6) were compared in ventricular myocardium from man, rats, rabbits, guinea pigs, pigeons and turtles. The most striking variation was in the activity of the ecto-5'-nucleotidase, which was 20 times less active in rabbit heart and 300 times less active in pigeon heart than in rat heart. The cytochemical distribution of ecto-5'-nucleotidase was also highly variable between species. 2. Adenosine formation was quantified in pigeon and rat ventricular myocardium in the presence of inhibitors of adenosine kinase and adenosine deaminase. 3. Both adenosine formation rates and the proportion of ATP catabolized to adenosine were greatest during the first 2 min of total ischaemia at 37 degrees C. Adenosine formation rates were 410 +/- 40 nmol/min per g wet wt. in pigeon hearts and 470 +/- 60 nmol/min per g wet wt. in rat hearts. Formation of adenosine accounted for 46% of ATP plus ADP broken down in pigeon hearts and 88% in rat hearts. 4. The data show that, in both pigeon and rat hearts, adenosine is the major catabolite of ATP in the early stages of normothermic myocardial ischaemia. The activity of ecto-5'-nucleotidase in pigeon ventricle (16 +/- 4 nmol/min per g wet wt.) was insufficient to account for adenosine formation, indicating the existence of an alternative catabolic pathway.     

Significance of adenosine metabolism of coronary smooth muscle cells

A detailed understanding of adenosine metabolism of vascular smooth muscle cells (VSMC) is highly desirable to critically evaluate possible autocrine effects of adenosine in this cell species. Therefore, this study quantified intra- and extracellular adenosine flux rates, the transmembrane concentration gradient, and the adenosine surface concentration in porcine VSMC and, for comparison, aortic endothelial cells (PAEC). Cell-covered microcarrier beads packed in a chromatography column were superfused with a HEPES buffer. With the use of specific inhibitors of adenosine kinase (iodotubericidine, 10 microM), adenosine deaminase [erythro-9-(2-hydroxy-3-nonyl)-adenine, 5 microM], ecto-5'-nucleotidase (alpha,beta-methylene-adenosine 5'-diphosphate, 50 microM), and adenosine membrane transport (n-nitrobenzylthioinosine, 1 microM), total production rates of 12.3 +/- 2.7 and 7.5 +/- 1.3 pmol x min(-1) x microl cell volume(-1) were obtained for VSMC and PAEC, respectively. Despite prevailing intracellular adenosine production (76 and 70% of total production, respectively), transmembrane concentration gradients under control conditions were directed toward the cytosol as a result of rapid intracellular adenosine rephosphorylation and continuous extracellular hydrolysis from 5'-AMP. Surface concentrations were approximately 18 nM in VSMC and PAEC under control conditions and increased to approximately 60 nM during partial inhibition of adenosine metabolism. Simultaneously, the transmembrane adenosine concentration gradient was reversed. We conclude that adenosine flux rates in VSMC and PAEC are quantitatively similar and that VSMC may influence the interstitial adenosine concentration under basal steady-state conditions.     

Expression of human ecto 5' nucleotidase in pig endothelial cells and its implication for adenosine production and xenotransplantation

Human endothelial activity of ecto-5'-nucleotidase (E5'N) is several times higher than in pig endothelial cells. This may have implication for xenotransplantation due to the role this enzyme plays in conversion of pro-inflammatory and pro-aggreggatory nucleotides into anti-inflammatory and antiaggregatory adenosine. We have shown in this study that human E5'N can be functionally expressed in pig endothelial cells leading to increased adenosine production from both extracellular AMP and ATP. We suggest that E5'N expression in transgenic pigs for xenotransplantation may help to prolong graft survival.     

Changes in cardiac nucleotide metabolism in Huntington's disease

ABSTRACT

Huntington's disease (HD) is a monogenic neurodegenerative disorder with a significant peripheral component to the disease pathology. This includes an HD-related cardiomyopathy, with an unknown pathological mechanism. In this study, we aimed to define changes in the metabolism of cardiac nucleotides using the well-established R6/2 mouse model. In particular, we focused on measuring the activity of enzymes that control ATP and other adenine nucleotides in the cardiac pool, including eNTPD, AMPD, e5′NT, ADA, and PNP. We employed HPLC to assay the activities of these enzymes by measuring the concentrations of adenine nucleotide catabolites in the hearts of symptomatic R6/2 mice. We found a reduced activity of AMPD (12.9 ± 1.9 nmol/min/mg protein in control; 7.5 ± 0.5 nmol/min/mg protein in R6/2) and e5′NT (11.9 ± 1.7 nmol/min/mg protein in control; 6.7 ± 0.7 nmol/min/mg protein in R6/2). Moreover, we detected an increased activity of ADA (1.3 ± 0.2 nmol/min/mg protein in control; 5.2 ± 0.5 nmol/min/mg protein in R6/2), while no changes in eNTPD and PNP activities were observed. Analysis of cardiac adenine nucleotide catabolite levels revealed an increased inosine level (0.7 ± 0.01 nmol/mg dry tissue in control; 2.7 ±0.8 nmol/mg dry tissue in R6/2) and a reduced concentration of cardiac adenosine (0.9 ± 0.2 nmol/mg dry tissue in control; 0.2 ± 0.08 nmol/mg dry tissue in R6/2). This study highlights a decreased rate of degradation of cardiac nucleotides in HD mouse model hearts, and an increased capacity for adenosine deamination, that may alter adenosine signaling.     

Neutrophil adhesion to xenogeneic endothelium via iC3b

Neutrophils are thought to play an important role in the pathogenesis of hyperacute rejection, a dramatic form of tissue injury caused by the reaction of antigraft antibodies with endothelial cells of an organ allograft or xenograft. We asked whether the interactions of human polymorphonuclear leucocytes (PMN) with xenogeneic endothelium might be promoted by the binding of natural anti-endothelial antibodies and complement by using porcine aortic endothelial cells (PAEC), human serum, and human PMN in an in vitro model of hyperacute rejection. Pretreatment of PAEC with 10% human serum followed by washing markedly increased PMN adhesion from 15.7 +/- 1.8% to 62.5 +/- 3.6% (p less than 0.001). Complement and anti-endothelial antibodies were necessary for the increase, because heat-inactivated serum or serum depleted of IgM did not significantly increase PMN adhesion to treated endothelium. The induction of increased PMN adhesion to PAEC by human serum was observed within 1 min. The essential role of complement was defined using complement-depleted serum. Ten percent C2-deficient serum did not increase PMN adhesion whereas 10% C5-depleted or 10% C8-depleted serum caused the same increase in PMN adhesion as observed with normal human serum. These results suggested that C3 might play a critical role in enhanced neutrophil adhesion. In fact, PAEC treated with 10% human serum for 15 min and incubated with an F(ab')2 antihuman C3 for 10 min completely abolished the enhanced adhesion. PAEC treated with 10% human serum or C5-depleted serum displayed fluorescence of iC3b whereas monolayers treated with heat-inactivated serum or C2-deficient serum were non-reactive. The enhanced PMN adhesion to serum-treated PAEC was mediated through neutrophil receptors binding iC3b because mAb directed against CD11b/CD18 inhibited the serum-enhanced adhesion of PMN. We conclude that PMN adhesion to endothelium can be significantly enhanced by the endothelial deposition of iC3b.     

A1 adenosine receptor overexpression attenuates ischemia-reperfusion-induced apoptosis and caspase 3 activity

We tested the hypothesis that myocardial ischemia-reperfusion (I/R)-induced apoptosis is attenuated in transgenic mice overexpressing cardiac A(1) adenosine receptors. Isolated hearts from transgenic (TG, n = 19) and wild-type (WT, n = 22) mice underwent 30 min of ischemia and 2 h of reperfusion, with evaluation of apoptosis, caspase 3 activity, function, and necrosis. I/R-induced apoptosis was attenuated in TG hearts. TG hearts had less I/R-induced apoptotic nuclei (0.88 +/- 0.10% vs. 4.22 +/- 0.24% terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cells in WT, P < 0.05), less DNA fragmentation (3.30 +/- 0.38-fold vs. 4.90 +/- 0.39-fold over control in WT, P < 0.05), and less I/R-induced caspase 3 activity (145 +/- 25% over nonischemic control vs. 234 +/- 31% in WT, P < 0.05). TG hearts also had improved recovery of function and less necrosis than WT hearts. In TG hearts pretreated with LY-294002 (3 microM) to evaluate the role of phosphosinositol-3-kinase in acute signaling, there was no change in the functional protection or apoptotic response to I/R. These data suggest that cardioprotection with transgenic overexpression of A(1) adenosine receptors involves attenuation of I/R-induced apoptosis that does not involve acute signaling through phosphoinositol-3-kinase.     

Kinetic characterization of adenosine deaminase activity in zebrafish (Danio rerio) brain

Adenosine deaminase (ADA; EC 3.5.4.4) activity is responsible for cleaving adenosine to inosine. In this study we described the biochemical properties of adenosine deamination in soluble and membrane fractions of zebrafish (Danio rerio) brain. The optimum pH for ADA activity was in the range of 6.0-7.0 in soluble fraction and reached 5.0 in brain membranes. A decrease of 31.3% on adenosine deamination in membranes was observed in the presence of 5 mM Zn(2+), which was prevented by 5 mM EDTA. The apparent K(m) values for adenosine deamination were 0.22+/-0.03 and 0.19+/-0.04 mM for soluble and membrane fractions, respectively. The apparent V(max) value for soluble ADA activity was 12.3+/-0.73 nmol NH(3) min(-1) mg(-1) of protein whereas V(max) value in brain membranes was 17.5+/-0.51 nmol NH(3) min(-1) mg(-1) of protein. Adenosine and 2'-deoxyadenosine were deaminated in higher rates when compared to guanine nucleosides in both fractions. Furthermore, a significant inhibition on adenosine deamination in both soluble and membrane fractions was observed in the presence of 0.1 mM of erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA). The presence of ADA activity in zebrafish brain may be important to regulate the adenosine/inosine levels in the CNS of this species.     

Functional characterization of Glu298Asp mutant human endothelial nitric oxide synthase purified from a yeast expression system.

The Glu298Asp polymorphism of human endothelial nitric oxide synthase (eNOS) has been reported to be associated with several cardiovascular diseases, including hypertension and myocardial infarction. Therefore, we investigated the effect of the Glu298Asp (E298D) mutation on the function of purified recombinant eNOS expressed in the yeast Pichia pastoris. Wild type (WT) and mutant exhibited comparable affinities for L-arginine (K(m) values 4.4+/-0.6 and 5.2+/-0.8 microM, respectively) and V(max) values (142+/-36 and 159+/-29 nmol of L-citrulline/mg min, respectively). The E298D mutation affected neither electron transfer through the reductase domain (measured as cytochrome c reduction) nor reductive O(2) activation (measured either as NADPH oxidation or as H(2)O(2) formation in the absence of L-arginine and tetrahydrobiopterin (BH4)). The mutant was activated by BH4 with an EC(50) of 0.24+/-0.04 microM, a value comparable to that obtained with WT eNOS (0.22+/-0.02 microM). Activation of the enzyme by Ca(2+) was not affected (EC(50)=0.50+/-0.04 and 0.49+/-0.02 microM for WT and E298D eNOS, respectively). Calmodulin (CaM) affinity, studied by radioligand binding using 125I-labeled CaM, revealed virtually identical K(D) (3.2+/-0.5 and 4.0+/-0.3nM) and B(max) (1.4+/-0.2 and 1.2+/-0.3 pmol/pmol subunit) values for WT and E298D eNOS, respectively. Furthermore, E298D eNOS did not differ from the WT enzyme with respect to heme and flavin content or the ability to form SDS-resistant dimers. To summarize, we obtained no evidence for altered enzyme function of the eNOS mutant that could explain endothelial dysfunction associated with the E298D polymorphism.     

Adenine incorporation in human and rat endothelium

Adenine (ADE) reutilisation is an important pathway of adenylate pool regeneration. Data on the rate of this process in different types of cells, its regulation and the importance of species differences is limited. In this study we evaluated adenine incorporation rate and the effect of metabolic factors on this process in human and rat endothelium and compared it to adenine phosphoribosyltransferase (APRT) activity. Microvascular endothelial cells from human (HE) and rat (RE) hearts and a transformed human microvascular endothelial cell line (HMEC-1) were investigated. The rate of adenine incorporation into the adenine nucleotide pool under control conditions was 3.1+/-0.3, 82.8+/-11.1 and 115.1+/-11.2 pmol/min per mg protein for HE, RE and HMEC-1, respectively. In the presence of 2.5 mM ribose or elevated inorganic phosphate concentration in the medium (4.8 mM), few changes were observed in all types of cells. In the presence of both ribose and high inorganic phosphate, the rate of adenine incorporation for RE and HMEC-1 was not significantly different from control, while in HE the rate of adenine incorporation into adenine nucleotides was increased by 75%. Activities of APRT in RE and HMEC-1 were 237.7+/-23.2 and 262.0+/-30.6 pmol/min per mg protein respectively while the activity in HE was markedly lower 48.7+/-3.0 pmol/min per mg protein. In conclusion, nucleotide synthesis from adenine seems to be a slow process in human cardiac microvascular endothelium but it is fast and efficient in rat heart microvascular endothelial cells. Low APRT activity in normal human endothelial cells seems to be the most likely mechanism for this. However, adenine incorporation rate and APRT activity could be greatly enhanced in human endothelium, as demonstrated in transformed cells.     

Direct oxidation of glutamate by mitochondria from porcine adrenal cortex

1. Mitochondria isolated from porcine adrenal cortex under State 3 conditions oxidized succinate with a rate of 47 +/- 4.48 na oxygen/min/mg/protein and with ADP:O ratio 0.98 +/- 0.09. In the presence of 15 microM deoxycorticosterone the rate of succinate oxidation was 36.8 +/- 3.08 na oxygen/min/mg/protein. 2. Under the same conditions the rate of glutamate oxidation was 22.8 +/- 2.21 and 16.8 +/- 0.65 na oxygen/min/mg/protein, respectively. ADP:O ratio was 1.45 +/- 0.14. 3. Introduction of trace amounts of malate into the mitochondria oxidizing glutamate only slightly increased the rate of O2 uptake. 4. The glutamate dehydrogenase activity in these mitochondria was 12.5 +/- 0.69 nmol/min/mg.     

Targeted deletion of A2A adenosine receptors attenuates the protective effects of myocardial postconditioning

Endogenous adenosine is an important ligand trigger for the cardioprotective effects of postconditioning (POC), yet it is unclear which adenosine receptor subtype is primarily responsible. To evaluate the role of A(2A) adenosine receptors in POC-induced protection, global ischemia-reperfusion was performed with and without POC in isolated wild-type (WT) and A(2A) adenosine receptor knockout (A(2A)KO) mouse hearts. Injury was measured in terms of postischemic functional recovery and release of cardiac troponin I (cTnI). Activation of protective signaling with POC was assessed by Akt and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation. In WT hearts, POC improved recovery of postischemic developed pressure in early (81.6 +/- 6.4% of preischemic baseline vs. 37.5 +/- 5.6% for non-POC WT at 1 min) and late (62.2 +/- 4.2% of baseline vs. 45.5 +/- 5.3% for non-POC WT at 30 min) reperfusion, reduced cTnI release by 37%, and doubled the phosphorylation of both Akt and ERK1/2. These beneficial effects of POC were blocked by treatment with the selective A(2A) adenosine receptor antagonist ZM-241385 during reperfusion. Postischemic functional recovery, cTnI release, and phosphorylation of Akt and ERK1/2 were not different between non-POC WT and A(2A)KO hearts. In A(2A)KO hearts, POC did not improve functional recovery, reduce cTnI release, nor increase phosphorylation of Akt or ERK1/2. Thus the protective effects of POC are attenuated by both selective A(2A) receptor antagonism and targeted deletion of the gene encoding A(2A) adenosine receptors. These observations support the conclusion that endogenous activation of A(2A) adenosine receptors is an essential trigger leading to the protective effects of POC in isolated murine hearts.     

Saturation mutagenesis of Burkholderia cepacia R34 2,4-dinitrotoluene dioxygenase at DntAc valine 350 for synthesizing nitrohydroquinone, methylhydroquinone, and methoxyhydroquinone

Saturation mutagenesis of the 2,4-dinitrotoluene dioxygenase (DDO) of Burkholderia cepacia R34 at position valine 350 of the DntAc alpha-subunit generated mutant V350F with significantly increased activity towards o-nitrophenol (47 times), m-nitrophenol (34 times), and o-methoxyphenol (174 times) as well as an expanded substrate range that now includes m-methoxyphenol, o-cresol, and m-cresol (wild-type DDO had no detectable activity for these substrates). Another mutant, V350M, also displays increased activity towards o-nitrophenol (20 times) and o-methoxyphenol (162 times) as well as novel activity towards o-cresol. Products were synthesized using whole Escherichia coli TG1 cells expressing the recombinant R34 dntA loci from pBS(Kan)R34, and the initial rates of product formation were determined at 1 mM substrate by reverse-phase high-pressure liquid chromatography. V350F produced both nitrohydroquinone at a rate of 0.75 +/- 0.15 nmol/min/mg of protein and 3-nitrocatechol at a rate of 0.069 +/- 0.001 nmol/min/mg of protein from o-nitrophenol, 4-nitrocatechol from m-nitrophenol at 0.29 +/- 0.02 nmol/min/mg of protein, methoxyhydroquinone from o-methoxyphenol at 2.5 +/- 0.6 nmol/min/mg of protein, methoxyhydroquinone from m-methoxyphenol at 0.55 +/- 0.02 nmol/min/mg of protein, both methylhydroquinone at 1.52 +/- 0.02 nmol/min/mg of protein and 2-hydroxybenzyl alcohol at 0.74 +/- 0.05 nmol/min/mg of protein from o-cresol, and methylhydroquinone at 0.43 +/- 0.1 nmol/min/mg of protein from m-cresol. V350M produced both nitrohydroquinone at a rate of 0.33 nmol/min/mg of protein and 3-nitrocatechol at 0.089 nmol/min/mg of protein from o-nitrophenol, methoxyhydroquinone from o-methoxyphenol at 2.4 nmol/min/mg of protein, methylhydroquinone at 1.97 nmol/min/mg of protein and 2-hydroxybenzyl alcohol at 0.11 nmol/min/mg of protein from o-cresol. The DDO variants V350F and V350M also exhibited 10-fold-enhanced activity towards naphthalene (8 +/- 2.6 nmol/min/mg of protein), forming (1R,2S)-cis-1,2-dihydro-1,2-dihydroxynaphthalene. Hence, mutagenesis of wild-type DDO through active-site engineering generated variants with relatively high rates toward a previously uncharacterized class of substituted phenols for the nitroarene dioxygenases; seven previously uncharacterized substrates were evaluated for wild-type DDO, and four novel monooxygenase-like products were found for the DDO variants V350F and V350M (methoxyhydroquinone, methylhydroquinone, 2-hydroxybenzyl alcohol, and 3-nitrocatechol).     

Human liver steroid sulphotransferase sulphates bile acids.

The sulphation of bile acids is an important pathway for the detoxification and elimination of bile acids during cholestatic liver disease. A dehydroepiandrosterone (DHEA) sulphotransferase has been purified from male and female human liver cytosol using DEAE-Sepharose CL-6B and adenosine 3',5'-diphosphate-agarose affinity chromatography [Falany, Vazquez & Kalb (1989) Biochem. J. 260, 641-646]. Results in the present paper show that the DHEA sulphotransferase, purified to homogeneity, is also reactive towards bile acids, including lithocholic acid and 6-hydroxylated bile acids, as well as 3-hydroxylated short-chain bile acids. The highest activity towards bile acids was observed with lithocholic acid (54.3 +/- 3.6 nmol/min per mg of protein); of the substrates tested, the lowest activity was detected with hyodeoxycholic acid (4.2 +/- 0.01 nmol/min per mg of protein). The apparent Km values for the enzyme are 1.5 +/- 0.31 microM for lithocholic acid and 4.2 +/- 0.73 microM for taurolithocholic acid. Lithocholic acid also competitively inhibits DHEA sulphation by the purified sulphotransferase (Ki 1.4 microM). No evidence was found for the formation of bile acid sulphates by sulphotransferases different from the DHEA sulphotransferase during purification work. The above results suggest that a single steroid sulphotransferase with broad specificity encompassing neutral steroids and bile acids exists in human liver.     

Early preconditioning prevents the loss of endothelial nitric oxide synthase and enhances its activity in the ischemic/reperfused rat heart

Cardiac ischemia may be responsible for either the loss of endothelial nitric oxide synthase (eNOS) or changes in its activity, both conditions leading to coronary dysfunction. We investigated whether early ischemic preconditioning was able to preserve eNOS protein expression and function in the ischemic/reperfused myocardium. Langendorff-perfused rat hearts were subjected to 20 min global ischemia, followed by 30 min reperfusion (I/R). A second group of hearts was treated as I/R, but preconditioned with three cycles of 5 min-ischemia/5 min-reperfusion (IP). Cardiac contractility markedly decreased in I/R, consistently with the rise of creatine kinase (CK) activity in the coronary effluent, whilst ischemic preconditioning significantly improved all functional parameters and reduced the release of CK. Western blot analysis revealed that the amount of eNOS protein decreased by 54.2% in I/R with respect to control (p < 0.01). On the other hand, NOS activity was not significantly reduced in I/R, as well as cGMP tissue levels, suggesting that a parallel compensatory stimulation of this enzymatic activity occurred during ischemia/reperfusion. Ischemic preconditioning completely prevented the loss of eNOS. Moreover, both NOS activity and cGMP tissue level were significantly higher (p < 0.05) in IP (12.7 +/- 0.93 pmol/min/mg prot and 58.1 +/- 12.2 fmol/mg prot, respectively) than I/R (7.34 +/- 2.01 pmol/min/mg prot and 21.4 +/- 4.13 fmol/mg prot, respectively). This suggest that early ischemic preconditioning may be useful to accelerate the complete recovery of endothelial function by preserving the level of cardiac eNOS and stimulating the basal production of nitric oxide.     

Presence of ectonucleotidases in cultured chromaffin cells: hydrolysis of extracellular adenine nucleotides

The granular ATP released from chromaffin cells during the secretory response can be hydrolyzed by ectonucleotidases that are present in the plasma membrane of these cells. The ecto-ATPase activity showed a Km for ATP of 250 +/- 18 microM and a VMAX value of 167 +/- 25 nmol/10(6) cells x min (1.67 mumol/mg protein x min) for cultured chromaffin cells, while the ecto-ADPase activity showed a Km value for ADP of 375 +/- 40 microM and a VMAX of 125 +/- 20 nmol/10(6) cells x min (1.25 mumol/mg protein x min). The ecto 5'-nucleotidase activity of cultured chromaffin cells was more specific for the purine nucleotides, AMP and IMP, than for the pirimidine nucleotides, CMP and TMP. The Km for AMP was 55 +/- 5 microM and the VMAX value was 4.3 +/- 0.8 nmol/10(6) cells x min (43 nmol/mg protein x min). The nonhydrolyzable analogs of ADP and ATP, alpha, beta-methylene-adenosine 5'-diphosphate and adenylyl-(beta, gamma-methylene)-diphosphonate were good inhibitors of ecto 5'-nucleotidase activity, the KI values being 73.3 +/- 3.5 nM and 193 +/- 29 nM, respectively. The phosphatidylinositol-specific phospholipase C released the ecto-5'-nucleotidase from the chromaffin cells in culture, thus suggesting an anchorage through phosphatidylinositol to plasma membranes. The presence of ectonucleotidases in chromaffin cells may permit the recycling of the extracellular ATP exocytotically released from these neural cells.     

Endocardial endothelium in the avascular frog heart: role for diffusion of NO in control of cardiac O2 consumption

We investigated the role of nitric oxide (NO) in the control of myocardial O(2) consumption in the hearts of female Xenopus frogs, which lack a coronary vascular endothelium and in which the endocardial endothelium is the only source of NO to regulate cardiac myocyte function. Hence, frogs are an ideal model in which to explore the role of diffusion of NO from the endocardial endothelium (EE) without vascular endothelial or cardiac cell NO production. In Xenopus hearts we examined the regulation of cardiac O(2) consumption in vitro at 25 degrees C and 37 degrees C. The NO-mediated control of O(2) consumption by bradykinin or carbachol was significantly (P < 0.05) lower at 25 degrees C (79 +/- 13 or 73 +/- 11 nmol/min) than at 37 degrees C (159 +/- 26 or 201 +/- 13 nmol/min). The response to the NO donor S-nitroso-N-acetyl penicillamine was also markedly lower at 25 degrees C (90 +/- 8 nmol/min) compared with 37 degrees C (218 +/- 15 nmol/min). When Triton X-100 was perfused into hearts, the inhibition of myocardial O(2) consumption by bradykinin (18 +/- 2 nmol/min) or carbachol (29 +/- 4 nmol/min) was abolished. Hematoxylin and eosin slides of Triton X-100-perfused heart tissue confirmed the absence of the EE. Although endothelial NO synthase protein levels were decreased to a variable degree in the Triton X-100-perfused heart, NO(2) production (indicating eNOS activity) decreased by >80%. It appears that the EE of the frog heart is the sole source of NO to regulate myocyte O(2) consumption. When these cells are removed, the ability of NO to regulate O(2) consumption is severely limited. Thus our results suggest that the EE produces enough NO, which diffuses from the EE to cardiac myocytes, to regulate myocardial O(2) consumption. Because of the close proximity of the EE to underlying myocytes, NO can diffuse over a distance and act as a messenger between the EE and the rest of the heart to control mitochondrial function and O(2) consumption.