We cultured the rabbit inner medullary collecting duct (IMCD) cells as monolayers on collagen-coated membrane filters, and investigated distribution of the P2Y receptors by analyzing nucleotide-induced short circuit current (Isc) responses. Exposure to different nucleotides of either the apical or basolateral surface of cell monolayers stimulated Isc. Dose-response relationship and cross-desensitization studies suggested that at least 3 distinct P2Y receptors are expressed asymmetrically on the apical and basolateral membranes. A P2Y2-like receptor, which responds to UTP and ATP, is expressed on both the apical and basolateral membranes. In addition, a uracil nucleotide receptor, which responds to UDP and UTP, but not ATP, is expressed predominantly on the apical membrane. In contrast, a P2Y1-like receptor, which responds to ADP and 2-methylthio-ATP, is expressed predominantly on the basolateral membrane. These nucleotides stimulated intracellular cAMP production with an asymmetrical profile, which was comparable to that in the stimulation of Isc. Our results suggest that the adenine and uracil nucleotides can interact with different P2Y nucleotide receptors that are expressed asymmetrically on the apical and basolateral membranes of the rabbit IMCD cells, and that both cAMP- and Ca2+-dependent signaling mechanisms underlie the stimulation of Isc.
Adenine ; Adenosine Diphosphate ; Adenosine Triphosphate ; Membranes ; Nucleotides ; Uracil ; Uracil Nucleotides ; Uridine Diphosphate ; Uridine Triphosphate

BACKGROUND: Adenosine 5'-tetraphosphate(ATPP), an endogenous nucleotide, is stored in cells and released into the extracellular space upon stimulation. Some of the biological responses to ATPP were reported, but characteristics of its receptor were not well known. Present study was conducted to investigate the effects of ATPP on mechanical contractility, resting membrane potential and action potential of rat left atrium. METHODS: Left atrium was isolated from Sprague-Dawley rat. Mechanical contraction induced by electrical field stimulation(EFS) was recorded on polygraph using force transducer. With glass microelectrodes(10 MOmega), potential difference across the membrane was measured and recorded on an oscilloscope and a polygraph. RESULTS: ATPP reduced the left atrial contractility with concentration-dependent manner. ATPP also hyperpolarized the resting membrane potential and decreased the action potential duration of the left atrial cell. Nucleotides other than ATPP, such as ATP, ADP, AMP and adenosine, have the same effect as ATPP. However, there is no difference among the nucleotides. Prior treatment of DPCPX, a P1-purinoceptor blocker, inhibited the ATPP-induced negative inotropism and changes of the membrane potential. But suramin, a nonselective P2-purinoceptor blocker, did not alter the effects of ATPP. alpha, beta methylene ADP and adenosine deaminase, which attenuates hydrolysis of adenine nucleotides and inactivates adenosine respectively, did not influence the effects of adenine nucleotides except for adenosine. CONCLUSION: ATPP reduced the mechanical contractility, hyperpolarized the resting membrance potential and decreased duration of action potential of rat left atrium. These effects were induced by ATPP directly, not by adenosine from hydrolyzed ATPP.
Action Potentials ; Adenine Nucleotides ; Adenosine Deaminase ; Adenosine Diphosphate ; Adenosine Triphosphate ; Adenosine* ; Animals ; Extracellular Space ; Glass ; Heart Atria ; Hydrolysis ; Membrane Potentials ; Membranes ; Muscle Contraction ; Nucleotides ; Rats ; Rats, Sprague-Dawley ; Receptors, Purinergic ; Suramin ; Transducers

Chinese hamster ovary (CHO) cells are the preferred host cells for the production of complex recombinant therapeutic proteins. Adenine phosphoribosyltransferase (APRT) is a key enzyme in the purine biosynthesis step that catalyzes the condensation of adenine with phosphoribosylate to form adenosine phosphate AMP. In this study, the gene editing technique was used to knock out the aprt gene in CHO cells. Subsequently, the biological properties of APRT-KO CHO cell lines were investigated. A control vector expressed an enhanced green fluorescent protein (EGFP) and an attenuation vector (containing an aprt-attenuated expression cassette and EGFP) were constructed and transfected into APRT-deficient and wild-type CHO cells, respectively. The stable transfected cell pools were subcultured for 60 generations and the mean fluorescence intensity of EGFP in the recombinant CHO cells was detected by flow cytometry to analyze the EGFP expression stability. PCR amplification and sequencing showed that the aprt gene in CHO cell was successfully knocked out. The obtained APRT-deficient CHO cell line had no significant difference from the wild-type CHO cells in terms of cell morphology, growth, proliferation, and doubling time. The transient expression results indicated that compared with the wild-type CHO cells, the expression of EGFP in the APRT-deficient CHO cells transfected with the control vector and the attenuation vector increased by 42%±6% and 56%±9%, respectively. Especially, the EGFP expression levels in APRT-deficient cells transfected with the attenuation vector were significantly higher than those in wild-type CHO cells (P < 0.05). The findings suggest that the APRT-deficient CHO cell line can significantly improve the long-term expression stability of recombinant proteins. This may provide an effective cell engineering strategy for establishing an efficient and stable CHO cell expression system.
Adenine/metabolism* ; Adenine Nucleotides ; Adenine Phosphoribosyltransferase/genetics* ; Adenosine Monophosphate ; Animals ; CHO Cells ; Cricetinae ; Cricetulus ; Recombinant Proteins/genetics*

Objective To determine the purine adenylate [adenosine triphosphate （ATP）, adenosine diphosphate （ADP）, adenosine monophosphate （AMP）] content in the muscles of both hind limbs of rats at different postmortem interval （PMI）, calculate the changes in the total adenine nucleotide （TAN） content and the adenylic-acid energy charge （AEC）, and explore their relationship with PMI. Methods Healthy rats were sacrificed by cervical dislocation and kept at 20 ℃. The muscles of their hind limbs were extracted at 0, 24, 48, 72, 96, 120, 144, and 168 h after death. Reversed-phase high performance liquid chromatography was used to determine the content of purine adenylates, the TAN and AEC of the muscles of the both hind limbs were calculated, and the related regression equations of their relationship with PMI were established. Results Within 168 h of death of rats, the trend of ATP change was different from ADP, and the content of AMP continuously increased. The TAN value gradually increased with the extension of PMI, and the AEC showed a downward trend within 168 h after death. Among them, the patterns of AEC changes with PMI were obvious, the correlation coefficient was high （ R²=0.903）, and the curve fitting relationship was good; the fitting relationship between ATP, ADP, AMP, TAN and PMI was poor （ R²=0.198-0.754）. Conclusion The postmortem change patterns of AEC provide new research ideas for PMI estimation in the forensic field.
Adenine Nucleotides ; Adenosine Monophosphate ; Animals ; Forensic Pathology ; Muscles ; Rats ; Rats, Sprague-Dawley ; Time Factors

The content of ATP, ADP, AMP, sodium phosphate and sodium pyrophosphate were determined by 31P NMR, the linear range of ATP, ADP and AMP were found to be 0.004-0.080 mol x L(-1), sodium phosphate and sodium pyrophosphate were 0.005-0.100 mol x L(-1). The RSD were 0.40%-1.30%, the recovery were 96.9% - 105.2%. The method has been applied to the determination of ATP injection. The impurities of ATP injection were ADP and sodium phosphate. The content of ATP is in line with the requirement of the pharmacopoeia. The results indicated that the method is of good reproducibility, high accuracy, rapid and simple operation, without pretreatment and interference of other elements, 31P NMR is a new and reliable method of analyzing ATP, ADP, AMP and phosphate.
Adenosine Diphosphate ; analysis ; Adenosine Monophosphate ; analysis ; Adenosine Triphosphate ; analysis ; Chemistry, Pharmaceutical ; methods ; Diphosphates ; analysis ; Magnetic Resonance Spectroscopy ; Perfusion ; Pharmaceutical Preparations ; analysis ; Phosphates ; analysis ; Quality Control ; Reproducibility of Results

Tomato extract has been shown to exert antiplatelet activity in vitro and to change platelet function ex vivo, but with limitations. In this study, antiplatelet activity of water soluble tomato concentrate (Fruitflow I) and dry water soluble tomato concentrate (Fruitflow II) was investigated using rat platelets. Aggregation was induced by collagen and adenosine diphosphate and granule-secretion, [Ca2+]i, thromboxane B2, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) levels were examined. The activation of integrin αIIbβ3 and phosphorylation of signaling molecules, including mitogen-activated protein kinase (MAPK) and PI3K/Akt, were investigated by flow cytometry and immunoblotting, respectively. Prothrombin time (PT) and activated partial thromboplastin time (aPTT) were examined. Moreover, in vivo thrombus weight was tested by an arteriovenous shunt model. Fruitflow I and Fruitflow II significantly inhibited agonist induced platelet aggregation, adenosine triphosphate and serotonin release, [Ca2+]i, and thromboxane B2 concentration, while having no effect on cAMP and cGMP levels. Integrin αIIbβ3 activation was also significantly decreased. Moreover, both concentrates reduced phosphorylation of MAPK pathway factors such as ERK, JNK, P38, and PI3K/Akt. In vivo thrombus formation was also inhibited. Taken together, these concentrates have the potential for ethnomedicinal applications to prevent cardiovascular ailments and can be used as functional foods.
Adenosine Diphosphate ; Adenosine Monophosphate ; Adenosine Triphosphate ; Animals ; Blood Platelets* ; Cardiovascular Diseases ; Collagen ; Flow Cytometry ; Functional Food ; Guanosine Monophosphate ; Immunoblotting ; In Vitro Techniques ; Lycopersicon esculentum* ; Partial Thromboplastin Time ; Phosphorylation ; Platelet Aggregation ; Protein Kinases* ; Prothrombin Time ; Rats ; Serotonin ; Thrombosis ; Thromboxane B2 ; Water*

Adenosine triphosphate (ATP) regeneration systems are essential for efficient biocatalytic phosphoryl transfer reactions. Polyphosphate kinase (PPK) is a versatile enzyme that can transfer phosphate groups among adenosine monophosphate (AMP), adenosine diphosphate (ADP), ATP, and polyphosphate (Poly P). Utilization of PPK is an attractive solution to address the problem of ATP regeneration due to its ability to use a variety of inexpensive and stable Poly P salts as phosphate group donors. This review comprehensively summarizes the structural characteristics and catalytic mechanisms of different types of PPKs, as well as the variations in enzyme activity, catalytic efficiency, stability, and coenzyme preference observed in PPKs from different sources. Moreover, recent advances in PPK-mediated ATP regeneration systems and protein engineering of wild-type PPK are summarized.
Adenosine Triphosphate/metabolism* ; Adenosine Monophosphate ; Polyphosphates/metabolism* ; Catalysis ; Regeneration

After renal injury, selective damage occurs in the proximal tubules as a result of inhibition of glycolysis. The molecular mechanism of damage is not known. Poly(ADP-ribose) polymerase (PARP) activation plays a critical role of proximal tubular cell death in several renal disorders. Here, we studied the role of PARP on glycolytic flux in pig kidney proximal tubule epithelial LLC-PK1 cells using XFp extracellular flux analysis. Poly(ADP-ribosyl)ation by PARP activation was increased approximately 2-fold by incubation of the cells in 10 mM glucose for 30 minutes, but treatment with the PARP inhibitor 3-aminobenzamide (3-AB) does-dependently prevented the glucose-induced PARP activation (approximately 14.4% decrease in 0.1 mM 3-AB-treated group and 36.7% decrease in 1 mM 3-AB-treated group). Treatment with 1 mM 3-AB significantly enhanced the glucose-mediated increase in the extracellular acidification rate (61.1±4.3 mpH/min vs. 126.8±6.2 mpH/min or approximately 2-fold) compared with treatment with vehicle, indicating that PARP inhibition increases only glycolytic activity during glycolytic flux including basal glycolysis, glycolytic activity, and glycolytic capacity in kidney proximal tubule epithelial cells. Glucose increased the activities of glycolytic enzymes including hexokinase, phosphoglucose isomerase, phosphofructokinase-1, glyceraldehyde-3-phosphate dehydrogenase, enolase, and pyruvate kinase in LLC-PK1 cells. Furthermore, PARP inhibition selectively augmented the activities of hexokinase (approximately 1.4-fold over vehicle group), phosphofructokinase-1 (approximately 1.6-fold over vehicle group), and glyceraldehyde-3-phosphate dehydrogenase (approximately 2.2-fold over vehicle group). In conclusion, these data suggest that PARP activation may regulate glycolytic activity via poly(ADP-ribosyl)ation of hexokinase, phosphofructokinase-1, and glyceraldehyde-3-phosphate dehydrogenase in kidney proximal tubule epithelial cells.
Animals ; Cell Death ; Epithelial Cells* ; Glucose ; Glucose-6-Phosphate Isomerase ; Glycolysis ; Hexokinase ; Kidney* ; LLC-PK1 Cells ; Oxidoreductases ; Phosphofructokinase-1 ; Phosphopyruvate Hydratase ; Poly Adenosine Diphosphate Ribose* ; Poly(ADP-ribose) Polymerases* ; Pyruvate Kinase ; Swine

OBJECTIVE: The content changes of energy substances in the cardiac muscle of rat killed by different manners were investigated to elucidate evidence that can be used to determine the modes of death and postmortem interval. METHODS: One hundred and eighty rats were randomly allocated into 3 groups and killed by bleeding, suffocating, and neck breaking, respectively. The contents of ATP, ADP, and AMP in the cardiac muscle of rats killed by the different manners at different death intervals (0, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 18, and 24 h) were measured by HPLC. RESULTS: There were significant differences observed in the contents of ATP and AMP in the rats' cardiac muscle in different groups at most of the intervals (P < 0.05) and at all of the intervals within the same group (P < 0.01), but no differences were found in the ADP contents in any of the group at most of the intervals. CONCLUSION The content changes of energy substances (ATP and AMP) in the cardiac muscle of dead rats may provide a basis for determination of the death manners and postmortem intervals.
Adenosine Diphosphate/metabolism* ; Adenosine Monophosphate/metabolism* ; Adenosine Triphosphate/metabolism* ; Animals ; Asphyxia/metabolism* ; Cause of Death ; Cervical Vertebrae/injuries* ; Chromatography, High Pressure Liquid ; Female ; Male ; Myocardium/pathology* ; Postmortem Changes ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Shock, Hemorrhagic/metabolism* ; Time Factors

BACKGROUND: Nucleoside transport inhibitor(NTI) Keeps AMP, ADP, ATP levels high in myocytes by inhibiting adenosine cataboilsm so that it may preserve the myocardial contractability during ischemia In this study we investigated the effects of cyclic AMP phosphodiesterase inhibor(C-AMP PDSI) and S-P-nitrobenzyl-6 -thioniosine(NBT; a sort of NIT) on myocadial preservation and changes of constituent enzyme. MATERIAL AND METHOD: Twenty-six isolated rabbit hearts were perfused with Krebs-Henseleit buffer solution for 20 minutes arrested for 20 minutes and ten reperfused for 30 minutes. The following four groups were prepared and hemodynamic changes coronary effluent lactate dehydrogenase (LDH) a-hydroxybutylic accid(a-HBD) levels and myocardial LDH creatine kinase-MB (CK-MB) adenosine deaminase(ADA) a-HBD levels and myocardial LDH creatine kinase-MB (CK-MB) adenosine deaminase(ADA) a-HBD levels were analysed before and after cardiac arest ; Group I(control) ; the heart was only perfused with K-H ; Group II ; the heart was perfused with K-H including C-AMP PDSI(Amrinone 25mg/L); Group III ; the heart was perfused with K-H including NBT(4.19mg/L) ; Group IV ; the heart was perfused with K-H including C-AMP PDSI + NBT. RESULT: Left venticular developed pressure(LVDP) at 10 minutes of the equilibrium was significantly higher in group III(72.1+/-5.3 mmHg p<0.01) and group III(72+/-5.6 mmHg P<0.025) as compared with group I (40.8+/-4.7mmHg) and LVDP at 20 minutes of the reperfusion was significantly higher in group II(74+/-5.3mmHg P<0.01) and group III(72+/-5.6mmHg p<0.025) as compared with group I (44.2+/-4.6mmHg). Percentage recovery of LVDP at the reperfusion was the highest in group II(123.3%) Percentage recovery of coronary flow at the equilibrium reperfusion were higher in group II(310%, 270%) group III(230%, 290%) group IV(310%, 280%) as compared with group I (100%) respectively. Myocadial LDH level was significant lower in group IV(33495+/-1802 IU/gm p<0.04) as compared with group I(48767+/-1421 IU/gm) Myocadial CK-MB level was significant higher in group II(74820+/-1421 IU/gm) compared with group I 45450+/-1737 IU/gm) Myocadial ADA level was significant higher group IV(1215+/-8 IU/gm p<0.05) compared with group I(125+/-15 IU/gm) but there was no significant difference between group I and group II ,III, IV in changes of coronary effluent LDH, a-HBD levels. CONCLUSIONS: C-AMP PDSI solely appears to have a better effect on myocardial preservation after ischemia than NBT but with no synergistic effect and it could keep CK-MB leve high in myocardial tissues.
Adenosine ; Adenosine Diphosphate ; Adenosine Triphosphate ; Creatine ; Cyclic AMP ; Heart ; Hemodynamics ; Ischemia ; L-Lactate Dehydrogenase ; Muscle Cells ; Myocardial Ischemia ; Myocardium* ; Reperfusion