Effects of alpha- and beta-adrenoceptor blockade on purine secretion induced by sympathetic nerve stimulation in the rat kidney

To characterize the effects of renal sympathetic nerve activation (RSNA) on renal purine secretion, 13 perfused rat kidneys were stimulated with periarterial electrodes at 7 Hz for 3 min, and purine secretion was determined by measuring with high-performance liquid chromatography purines in the renal venous perfusate 1 min before and during the last minute of RSNA. RSNA significantly increased renal perfusion pressure and significantly increased the secretion of adenosine and adenosine metabolites (inosine, hypoxanthine, and xanthine) by 2- to 5-fold. To investigate the participation of alpha- and beta-adrenoceptors in this response, four groups of perfused kidneys (n = 5/group) were pretreated with either vehicle, prazosin (alpha1-adrenoceptor antagonist; 0.03 microM), phentolamine (alpha1/2-adrenoceptor antagonist; 3 microM), or propranolol (beta1/2-adrenoceptor antagonist; 0.1 microM), and purine secretion was measured before and during RSNA at 1, 3, 5, 7, and 9 Hz. Prazosin, phentolamine, and propranolol abolished the RSNA-induced increase in the secretion of adenosine, inosine, hypoxanthine, and xanthine. In contrast, prazosin and phentolamine nearly abolished, whereas propranolol only slightly reduced, renal vascular responses to RSNA. Our results indicate that RSNA increases renal purine secretion via a mechanism that requires both alpha- and beta-adrenoceptors. It is well known that in the kidney adenosine activates renal afferent nerves, enhances renovascular responses to norepinephrine and angiotensin II, and increases sodium reabsorption; therefore, RSNA-induced adenosine production may contribute to the hypertensive effects of RSNA. Moreover, the antihypertensive effects of beta-adrenoceptor antagonists may in part be due to inhibition of RSNA-induced renal adenosine production.     

Screening a peptidyl database for potential ligands to proteins with side-chain flexibility

OBJECTIVE: Microdialysis and 31P-NMR spectroscopy were used to test opposing hypotheses that ischemic preconditioning inhibits adenine nucleotide degradation and purine efflux, or that preconditioning activates cardiovascular adenosine formation to provide enhanced cardioprotection. METHODS: 31P-NMR spectra and matching interstitial fluid (ISF) or venous effluent samples were obtained from Langendorff perfused rat hearts. Control hearts (n = 9) underwent 30 min of global normothermic ischemia and 30 min reperfusion. Preconditioned hearts (n = 6) were subjected to a 5 min ischemic episode and 10 min reflow prior to 30 min ischemia and 30 min reperfusion. Effects of repetitive ischemia-reperfusion (3 x 5 min ischemic episodes) on adenosine levels and energy metabolism were also assessed (n = 8). RESULTS: Preconditioning improved post-ischemic recovery of heart rate x left ventricular developed pressure (71 +/- 5 vs 43 +/- 8%, P < 0.05) and end-diastolic pressure (14 +/- 3 vs 29 +/- 4 mmHg, P < 0.05) compared with control hearts, respectively. Preconditioning did not alter intracellular ATP, phosphocreatine (PCr), inorganic phosphate (Pi), H+ or free Mg2+ during global ischemia, but improved recoveries of PCr, Pi, and delta GATP on reperfusion. ISF adenosine increased more than 20-fold during 30 min ischemia. The 5 min preconditioning episode increased ISF adenosine 3-fold, and reduced ISF adenosine and inosine during subsequent prolonged ischemia by up to 75%. Venous purine levels during reperfusion were also reduced by preconditioning. Accumulation of adenosine in ISF and venous effluent during repetitive ischemia was progressively reduced despite comparable changes in substrate for adenosine formation via 5'-nucleotidase, (5'-AMP), and in allosteric modulators of this enzyme (Mg2+, H+, Pi, ADP, ATP). CONCLUSIONS: (i) Ischemic preconditioning reduces interstitial and vascular adenosine levels during ischemia-reperfusion, (ii) reduced ISF adenosine during ischemia is not due to reduced ischemic depletion of adenine nucleotides in preconditioned rat hearts, (iii) preconditioning may inhibit adenosine formation via 5'-nucleotidase in ischemic rat hearts, and (iv) improved functional recovery with preconditioning is unrelated to metabolic/bioenergetic changes during the ischemic insult, but may be related to improved post-ischemic recovery of [Pi] and delta GATP in this model.     

Studies of 5'-nucleotidase in the perfused rat heart. Including measurements of the enzyme in perfused skeletal muscle and liver

Perfused rat hearts catalyze the hydrolysis of AMP added to the perfusion fluid at a rate of 35 mumol/g dry weight/min. The activity is specific for 5'-nucleoside monophosphates, little activity being observed with 2' and 3'-AMP. The enzyme exhibits Michaelis-Menten kinetics in situ and is inhibited competitively by adenosine-5'-alpha, beta-methylene diphosphonate (Ki = 13 muM). This, as well as the nucleotide specificity, confirms that the hydrolysis is catalyzed by 5'-nucleotidase. The maximum activity of 5'-nucleotidase in perfused hearts is equal to or greater than that found in heart homogenates; thus, all of the enzyme is accessible to AMP added externally. Hydrolysis of endogenous AMP was studied in the perfused heart. Under aerobic conditions hearts contain very low amounts of purine nucleosides, and little or no nucleoside is found in the effluent perfusate. Under anaerobic conditions hearts accumulate adenosine, inosine, and hypoxanthine and release all three substances into the perfusate. Hydrolysis of externally added AMP was also observed in perfused skeletal muscle and liver, at rates of 10 and 17 mumol/g dry weight/min, respectively.     

Upstream stimulatory factor 2 activates the mammalian F1F0 ATP synthase alpha-subunit gene through an initiator element

Adenosine is a putative neuroprotectant in ischemia, but its role after traumatic brain injury (TBI) is not clear. Metabolites of adenosine, particularly inosine and hypoxanthine, are markers of ischemia and energy failure. Adenosine triphosphate (ATP) breakdown early after injury and metabolism of cyclic adenosine monophosphate (cAMP) are potential sources of adenosine. Further delineation of the magnitude, location, time course, and source of production of adenosine after TBI is needed. We measured adenosine, inosine, and hypoxanthine in brain interstitial fluid after controlled cortical impact (CCI) in the rat. Rats (n = 15) were prepared for TBI induced by CCI. A microdialysis probe was placed in the cortex, and samples were collected every 10 min. After 3 h of equilibration, the catheter was removed, CCI was performed (4 m/sec, depth 2.5 mm), and the catheter was replaced. In the shams, the catheter was removed and replaced without CCI. The injury group included rats (n = 10) subjected to CCI. Within the injury group, the microdialysis probe was placed in the center of the eventual contusion (center, n = 5) or in the penumbral region (penumbra, n = 5). Purine metabolites were measured using ultraviolet-based high-pressure liquid chromatography. Adenosine, inosine, and hypoxanthine were dramatically increased after injury (61-fold, 37-fold, and 16-fold, respectively sham, all p < 0.05, two-way analysis of variance for repeated measures). No changes in cAMP were observed (p = 0.62 vs. sham). Adenosine peaked in the first 20 min and returned to near baseline 40 min, whereas inosine and hypoxanthine peaked at 30 min and remained increased for 40 min after CCI. Interstitial brain adenosine, inosine, and hypoxanthine were increased early after CCI in rats in the contusion and penumbra. ATP breakdown is a potential source of adenosine in this early period while metabolism of cAMP does not appear to play a role. Confirmation of these data in humans may suggest new strategies targeting this important metabolic pathway.     

In vivo and in vitro developmental toxicity in LPS-induced zinc-deficient rabbits

Adenosine, produced from the decomposition of adenosine triphosphate, is believed to provide protective effects during ischemia. On the other hand, adenosine metabolites may serve as precursors for oxygen free radical formation. The time course of formation of adenosine and its purine metabolites was studied during retinal ischemia in rats. Concentrations of adenosine and its purine nucleoside metabolites inosine, hypoxanthine, and xanthine in the retina-choroid of ketamine/xylazine-anesthetized rats were measured during retinal ischemia using high performance liquid chromatography. Quantitative measurements were made possible in the small tissue mass through the use of internal standards. Ischemia was induced by ligation of the central retinal artery. In each rat, one eye was ischemic while the other served as a non-ischemic control. Eyes were frozen in situ at 1, 5, 10, 20, 30, 60, and 120 min of ischemia. The retina-choroid was then removed from the frozen eyes and analysed. Significant increases in the concentrations of adenosine, inosine, and hypoxanthine in ischemic compared to control retina-choroid were detectable within 1 to 5 min of the onset of ischemia, and within 10 min for xanthine. Increase in adenosine concentration in ischemic relative to control retina-choroid plateaued at 30 min of ischemia, while inosine and hypoxanthine concentrations increased continuously. The increase in xanthine concentration was exponential throughout the measurement period. This study documented the time-related changes in purine nucleoside concentration during ischemia. Prolonged ischemia results in ongoing production of xanthine, which by serving as a precursor for oxygen free radical formation, could be a pathogenic factor in prolonged retinal ischemia.     

The use of computed tomography in stereotaxic operations in dyskinesia patients

The pool of free purine derivatives and activities of the key enzymes of purine metabolism (adenosine deaminase, purine nucleoside phosphorylase, and 5'-nucleotidase) in lymphocytes, erythrocytes, and epidermis homogenates were measured in 20 normal subjects and 15 patients with psoriasis by high-performance liquid chromatography. The levels of AMP, GMP, and IMP purine monophosphates are decreased in the epidermis and red cells of psoriasis patients, whereas the final products of hypoxanthine, xanthine, and uric acid metabolism are accumulating, and the activities of ADA and PNP are increased double in the skin, all this indicating purine derivatives catabolism.     

Regulation of purine nucleotide synthesis. Effects of inosine on normal and hypoxantine-guanine phosphoribosyltransferase-deficient fibroblasts

Incubation of normal and hypoxanthine-guanine phosphoribosyltransferase-deficient (mutant) human fibroblasts with inosine results in increased intracellular concentration of 5-phosphoribosyl 1-pyrophosphate (PP-ribose-P). The magnitude of this increase is dependent on the concentration of the nucleoside and results from donation of the ribose moiety of inosine to the ribosyl phosphate moiety of PP-ribose-P through ribose phosphate intermediates. During incubation, rates of purine nucleotide synthesis de novo, estimated by incorporation of (14C) formate into formylglycinamide ribotide, are diminished in both normal and mutant cells: 5 mM inosine inhibits purine synthesis by 60-80% in normal cells and 2-20% in hypoxanthine-guanine phosphoribosyltransferase-deficient cells. The rates of purine synthesis in both normal and mutant cells are increased, however, during incubation with methylene blue at concentrations (50-100 muM) which result in more modest increases in ribose 5-phosphate and PP-ribose-P concentrations than are observed with inosine. Saturation of the PP-ribose-P amidotransferase reaction by PP-ribose-P does not appear, therefore, to explain the failure of increased PP-ribose-P concentration to stimulate the rate of purine synthesis in either type of fibroblast during incubation with inosine. Although the dissociation between PP-ribose-P concentration and the rate of purine nucleotide synthesis in normal fibroblasts incubated with inosine may be explained at least in part by an accompanying increase in intracellular concentrations of purine nucleotide feedback inhibitors, purine nucleotide concentrations are unchanged in mutant cells during incubation with inosine; these cells, in addition, show minimal (less than 3% of normal) incorporation of labeled hypoxanthine or the hypoxanthine moiety of inosine into purine nucleotides. The effect of inosine on purine synthesis de novo in hypoxanthine-guanine phosphoribosyltransferase-deficient fibroblasts is not explained in full by consideration of the concentrations of purine nucleotides and of PP-ribose-P, the factors frequently invoked as antagonistic regulators controlling the rate of this process.     

The inhibitory effect of angiotensin II on stimulus-induced release of cAMP is augmented in the genetically hypertensive rat kidney

In the present study, with isolated perfused kidneys, we evaluated whether angiotensin II (Ang II) inhibits stimulus-induced release of adenosine 3',5'-cyclic monophosphate (cAMP) and whether this effect is augmented in spontaneously hypertensive rats (SHR). The basal release of cAMP (in venous effluent) in the presence of captopril (1 mumol/l) and a phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (10 mumol/l), was significantly (P < .05) higher in the SHR (n = 20) than in Wistar-Kyoto (WKY) kidneys (n = 18) although perfusion pressures were not significantly different in the two strains. Isoproterenol infusions (ISO; 0.3, 1 and 3 mumol/l) significantly and similarly increased cAMP release in both WKY (n = 5; P < .01) and SHR (n = 6; P < .01) kidneys. A time-related attenuation of the cAMP response to ISO in both strains was observed in these experiments. In control experiments, Ang II (3 and 10 ng/min), by itself, did not significantly alter basal cAMP release in either strain but raised perfusion pressure in both SHR and WKY kidneys. In a separate set of experiments, Ang II significantly (3 ng/min: P < .05; 10 ng/min: P < .01) inhibited ISO-induced increases in release of cAMP from SHR kidneys (n = 8), whereas cAMP release in response to ISO in WKY kidneys (n = 8) was not affected by Ang II (3 and 10 ng/min). In the same experiments, ISO produced small but significant decreases in perfusion pressure in WKY (P < .01) but not in SHR. These data clearly and directly demonstrate that ISO-induced increases in cAMP in the renal vasculature are similar in SHR and WKY rats; however, Ang II exerts a much greater negative influence on the ISO-induced increases in cAMP levels in the renal vasculature of SHR. The augmented inhibition of stimulus-induced cAMP release may be associated with an increased renovascular responsiveness to Ang II in SHR.     

Comparison of interstitial fluid and coronary venous adenosine levels in in vivo porcine myocardium

There are numerous reports of interstitial fluid (ISF) and coronary venous adenosine measurements in isolated perfused hearts. This study was designed to simultaneously compare ISF and coronary venous adenosine concentrations during various interventions in in vivo porcine myocardium. In anesthetized, open-chest pigs, ISF adenosine, inosine, and hypoxanthine were sampled with cardiac microdialysis. Coronary sinus or venous purines were sampled with a metabolism-stop solution. During basal conditions, ISF adenosine was greater than coronary venous adenosine, but vascular inosine and hypoxanthine were greater than corresponding ISF levels. Dobutamine (20 micrograms/kg/min, i.v.) and systemic hypoxia produced three- and two-fold increases in ISF adenosine, but had no significant effect on coronary sinus adenosine concentration. Hypoxia, but not dobutamine, increased coronary sinus total purines 50%. In contrast to these interventions, intracoronary adenosine infusion (0.5-50 micrograms/kg/min) was associated with significantly greater coronary venous adenosine concentrations than ISF levels. Only during a coronary artery occlusion/reperfusion protocol were ISF and coronary venous adenosine concentrations comparable. The results of this study thus provide in vivo evidence of the powerful endothelial and red blood cell metabolic barriers to both exogenous and endogenous adenosine. These results also illustrate the differences in adenosine concentrations in the ISF and vascular spaces.     

NO and cGMP facilitate adenosine production in rat hearts via activation of ecto-5'-nucleotidase

We examined whether nitric oxide (NO), a possible cardioprotective substance, can increase the production of interstitial adenosine in the ventricular myocardium. A flexibly mounted microdialysis technique was used to measure the concentration of interstitial adenosine and to assess the activity of ecto-5'-nucleotidase in in vivo rat hearts. The microdialysis probe was implanted in the left ventricular myocardium of anesthetized rats and perfused with Tyrode solution containing adenosine 5'-monophosphate (AMP) at a rate of 1.0 microl min-1. The concentration of adenosine in the effluent (dialysate) was measured by high-performance liquid chromatography. Dialysate adenosine obtained during perfusion with the AMP-containing solution through the probe originated from the hydrolysis of AMP by endogenous ecto-5'-nucleotidase, and the level of adenosine reflected the activity of ecto-5'-nucleotidase in the tissue. S-Nitroso-N-acetylpenicillamine (SNAP, 0.3-3 mM), an NO donor, increased the dialysate adenosine measured in the presence of AMP (100 microM) in a concentration-dependent manner. However, in the presence of an NO-oxidizing agent, 2-(4-carboxyphenyl-4,4,5, 5-tetramethylimidazoline)-1-oxyl 3-oxide (carboxy-PTIO, 1 mM), the effect of SNAP was abolished. Another NO donor, (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (FK409, 1 mM) also increased adenosine production. 8-Bromo-cGMP (0.1-3 mM), a membrane-permeable cGMP analogue and a potent activator of cGMP-dependent protein kinase, increased the level of AMP-primed dialysate adenosine in a concentration-dependent manner. These results suggest that NO facilitates the production of interstitial adenosine in rat hearts in situ, via cGMP-mediated activation of ecto-5'-nucleotidase.     

Weight loss education: the challenge of a new paradigm

The functional effects of adenosine 5'-triphosphate (ATP), uridine 5'-triphosphate (UTP), adenosine 5'-tetraphosphate (AP4) and the diadenosine polyphosphates P1,P3-diadenosine triphosphate (Ap3A), P1,P4-diadenosine tetraphosphate (Ap4A) and P1,P5-diadenosine pentaphosphate (Ap5A) were studied in two isolated smooth muscle preparations thought to contain P2Y (P2Y1) receptors, the guinea-pig taenia caeci (which relaxes to ATP) and the rat colon muscularis mucosae (which contracts to ATP). In addition, the breakdown of these compounds by the rat colon muscularis mucosae was investigated by high pressure liquid chromatography. In the guinea-pig taenia caeci all the purine nucleotides caused relaxation with a potency order of Ap3A=Ap4A> ATP>AP4=Ap5A, and these relaxations were antagonised by suramin with apparent pA2 values in the region of 5, consistent with activation of a P2Y1 receptor. In the rat colon muscularis mucosae the nucleotides caused contraction with a potency order of Ap3A = Ap4A>ATP=AP4 =Ap5A >UTP. However, while suramin (100 microM) inhibited responses to ATP and UTP at all concentrations of agonist, it only inhibited contractions induced by the higher concentrations of AP4, Ap3A and Ap4A and had little effect on contractions induced by Ap5A. A higher concentration of suramin (1 mM) enhanced contractions induced by ATP but greatly inhibited those induced by UTP and had no effect on responses to the other agonists. The A1 adenosine receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 10 nM) had no effect on responses to ATP or UTP but inhibited responses to Ap3A, Ap4A, Ap5A and AP4. A combination of suramin (1 mM) and DPCPX (10 nM) almost abolished responses to all the agonists. ATP and UTP were rapidly degraded by the rat colon muscularis mucosae while AP4, Ap3A, Ap4A and Ap5A were degraded more slowly, and the major product detected after breakdown of the purine nucleotides was inosine rather than adenosine. The breakdown of all the nucleotides was inhibited by suramin (1 mM), although this inhibition did not achieve statistical significance in the case of ATP. These results show that while the diadenosine polyphosphates appear to act as P2 agonists in the taenia caeci, in the rat colon muscularis mucosae their major action is via adenosine A1 receptors rather than via P2 receptors. In addition, although they are more stable than ATP or UTP, their action in this tissue is clearly affected by their degradation which complicates the effects of suramin.     

Reduced thiopurine methyltransferase activity and development of side effects of azathioprine treatment in patients with rheumatoid arthritis

OBJECTIVE: To investigate thiopurine enzyme activities for their possible value in predicting the development of azathioprine (AZA)-related toxicity in patients with rheumatoid arthritis (RA). METHODS: Patients with longstanding RA (n = 33) were enrolled in a study of treatment with AZA. Before the initiation of AZA treatment and at months 1 and 6 of treatment, we measured activities of the purine key enzymes hypoxanthine guanine phosphoribosyltransferase, 5'-nucleotidase, purine nucleoside phosphorylase, and thiopurine methyltransferase (TPMT). Controls included patients with early RA (n = 24) and healthy volunteers (n = 42). RESULTS: Fourteen of the 33 patients rapidly developed severe side effects, most frequently gastrointestinal (GI) intolerance. Compared with the other groups, the group with adverse effects had significantly lower TPMT activities (P = 0.004). Seven of 8 patients with reduced ("intermediate") baseline TPMT levels developed toxicity, resulting in a significant relationship (P = 0.005) between toxicity and "intermediate" TPMT activity. Compared with "high" activity, baseline intermediate TPMT activity gave a relative risk of 3.1 (95% confidence interval 1.6-6.2) for the development of severe toxicity with AZA treatment. CONCLUSION: In RA patients, inherited intermediate TPMT activity seems predictive for the development of severe side effects of AZA. Clinicians should consider measuring TPMT prior to treatment initiation to improve the safety of AZA use. We hypothesize that GI intolerance may also be related to a thiopurine metabolic imbalance.     

Extracellular adenosine 5'-triphosphate induces Ca2+ efflux from freshly isolated adult rat cardiomyocytes: possible involvement of Na+/Ca2+ exchange mechanism

In the present study, we examined the effect of extracellular adenosine 5'-triphosphate (ATP) on Ca2+ efflux from freshly isolated adult rat cardiomyocytes. ATP at 1 mM caused a release of 3.6+/-0.08% of the total cellular content. The 45Ca2+ efflux from the cells was also stimulated by adenosine-5'-O-(3-thiotriphosphate) (ATP-gamma s), alpha, beta-methylene-ATP and adenosine 5'-diphosphate (ADP), but not by adenosine 5'-monophosphate (AMP) or adenosine. The effect of ATP was inhibited by a known purinergic P2-receptor antagonist, but not by a P1-receptor antagonist. From these results, it is conceivable that the effect of ATP on Ca2+ efflux from cardiomyocytes is mediated through P2-purinoceptors. It was also observed that ATP caused a rise in [Ca2+]i to almost 200 nM. The ATP-stimulated 45Ca2+ efflux was not affected by removal of extracellular Ca2+, but was dependent on the presence of extracellular Na+. Moreover, ATP caused a 22Na+ influx into the cells of about 2.0-fold over the basal value. These result suggest that ATP stimulates extracellular Na+-dependent 45Ca2+ efflux from freshly isolated adult rat cardiomyocytes, probably through its stimulatory effect on plasma membrane P2-purinoceptors which may couple to Na+/Ca2+ exchange.     

A comparison of AMP degradation in the perfused rat heart during 2-deoxy-D-glucose perfusion and anoxia. Part I: The release of adenosine and inosine

AMP degradation is studied in two models of the Langendorff-perfused rat heart which generate a large release of purines: the 2-deoxy-D-glucose (2DG)-perfused heart and the anoxic heart. In the 2DG model, mitochondrial energy generation is quasi-normal, despite a very low ATP concentration. Furthermore, inorganic phosphate (Pi) concentration is low, an important difference with anoxia where Pi is very high, up to 82 mM. Coronary release of purines is measured by high performance liquid chromatography, and myocardial metabolite content by 31P nuclear magnetic resonance spectroscopy. In the 2DG-perfused hearts with glucose or acetate, the purine release consists nearly exclusively of inosine [up to 130 nmol/(min x gww)] while adenosine is less than 1 nmol/(min x gww). A possible interpretation is that AMP degradation proceeds mainly through deamination to inosine monophosphate by AMP deaminase (the IMP pathway). In contrast, the purine release in anoxia (100% N2) contains comparable quantities of adenosine and inosine [respectively 30 and 20 nmol/(min x gww)], indicating that part of AMP is dephosphorylated directly to adenosine. Comparison with the 2DG model suggests that the release of adenosine in the anoxic heart is a result of inhibition of AMP deaminase by Pi.     

Fetal plasma hypoxanthine level in growth-retarded fetuses before labor

Hypoxanthine is one of the purine nucleotides and is presumed to accumulate during hypoxia and acidemia. It remains uncertain, however, whether plasma hypoxanthine concentration is a useful indicator of fetal asphyxia; and its relationship to other markers of fetal physiologic state is not clearly defined. The aim of this study was to evaluate whether the level of fetal plasma hypoxanthine is correlated with fetal hypoxia and acidosis in growth-retarded fetuses before the onset of labor. Cordocentesis was performed in 34 growth-retarded fetuses at 31-35 weeks' gestation for the measurement of umbilical venous plasma concentrations of hypoxanthine, hemoglobin and lactate concentrations, blood gases, and base deficit. Umbilical venous plasma hypoxanthine concentration was found to be increased significantly, in parallel with the degree of acidosis (r = -0.74, P < 0.05) and base deficit (r = -0.41, P < 0.05), but not to bear a significant relationship to the degree of hypoxemia or other measured variables. We conclude that increases in the plasma concentration of hypoxanthine may reflect an impaired physiological state in growth-retarded fetuses before labor.     

Global disparities in health and human rights: a critical commentary

We and others have shown that an increased extracellular concentration of adenosine mediates the antiinflammatory effects of methotrexate and sulfasalazine both in vitro and in vivo, but the mechanism by which these drugs increase extracellular adenosine remains unclear. The results of the experiments reported here provide three distinct lines of evidence that adenosine results from the ecto-5'-nucleotidase- mediated conversion of adenine nucleotides to adenosine. First, pretreatment of a human microvascular endothelial cell line (HMEC-1) with methotrexate increases extracellular adenosine after exposure of the pretreated cells to activated neutrophils; the ecto-5'-nucleotidase inhibitor alpha, beta-methylene adenosine-5'-diphosphate (APCP) abrogates completely the increase in extracellular adenosine. Second, there is no methotrexate-mediated increase in extracellular adenosine concentration in the supernate of cells deficient in ecto-5'-nucleotidase, but there is a marked increase in extracellular adenosine concentration in the supernates of these cells after transfection and surface expression of the enzyme. Finally, as we have shown previously, adenosine mediates the antiinflammatory effects of methotrexate and sulfasalazine in the murine air pouch model of inflammation, and injection of APCP, the ecto-5'-nucleotidase inhibitor, abrogates completely the increase in adenosine and the decrement in inflammation in this in vivo model. These results not only show that ecto-5'-nucleotidase activity is a critical mediator of methotrexate- and sulfasalazine-induced antiinflammatory activity in vitro and in vivo but also indicate that adenine nucleotides, released from cells, are the source of extracellular adenosine.     

A study of the 30 minutes following reperfusion after crystalloid and cold blood cardioplegia by enzymatic and metabolic analysis of coronary blood flow

Post-ischemic reperfusion phenomena were studied in two methods of myocardial protection: crystalloid cardioplegia (St Thomas n(o) 2) and cold blood cardioplegia (Buckherg) during cardiopulmonary bypass for human myocardial revascularisation. Myocardial protection was assessed on the course of hemodynamic parameters, reperfusion arrhythmias and biochemical analysis of the coronary flow after cross-clamp removal: creatine phosphokinase (CPK-MB) and nucleotide adenine metabolites (adenosine, inosine, hypoxanthine, xanthine and uric acid). The study was performed in two groups of 14 patients. Hemodynamic conditions were similar in both groups during reperfusion in order to avoid different coronary flow. Under these conditions, myocardial protection by cold blood cardioplegia reduced reperfusion arrhythmias, and resulted in a loss of CPK-MB release. Furthermore, cold blood cardioplegia provided protection of myocardial energy metabolism by reducing the loss of metabolites, purine bases and oxypurine bases into the coronary sinus. Our results also show that hypoxanthine is probably the final product of ATP degradation in human myocardial tissue.     

The effect of selective phosphodiesterase inhibitors on plasma insulin concentrations and insulin secretion in vitro in the rat

We have examined in rats the effects of Org 9935 (4,5-dihydro-6-(5,6-dimethoxy-benzo[b]-thien-2-yl)-methyl-1-(2H)-p yridazinone), a selective inhibitor of type 3 phosphodiesterase (phosphodiesterase 3) and Org 30029 (N-hydroxy-5,6-dimethoxy-benzo[b]-thiophene-2-carboximidamide HCl), an inhibitor of phosphodiesterase 3/4 on rat plasma insulin and glucose concentrations in pentobarbitone-anaesthetised rats and on insulin secretion by rat isolated islets. We have also compared their effects on islet phosphodiesterase activity. Org 9935 (0.1 and 1.0 mg kg(-1) i.v. 15 min previously) dose dependently elevated fasting and post-glucose (0.25 g kg(-1) i.v.) plasma insulin concentrations. Org 30029 in a dose of 10 mg kg(-1), but not 1 mg kg(-1), also increased plasma insulin concentrations. Neither drug modified either fasting or post-glucose plasma glucose concentrations. Each drug augmented glucose-induced insulin release by rat isolated islets in a static incubation system, with approximate EC50 values of 1.5 microM for Org 9935 and 20 microM for Org 30029. Phosphodiesterase activity, in both supernatant and pellet fractions of islet homogenates, was inhibited concentration dependently by each drug. Although the shape of the concentration-inhibition curve for Org 30029 precluded estimation of an IC50 value, this drug was clearly much less potent than Org 9935 (IC50 about 50 nM) in inhibiting islet phosphodiesterase activity. We conclude that the increase in plasma insulin produced by each drug is a consequence of augmented insulin secretion, probably secondary to inhibition of phosphodiesterase 3 in the islet beta cell, with a resultant elevation in cAMP. The failure of the drugs to modify plasma glucose may be due to concomitant inhibition of cAMP phosphodiesterase in liver and adipose tissue.     

DNA vaccines for bacterial infections

The uptake and transportation of purine and pyrimidine based nucleosides by trophozoites of axenically grown Entamoeba histolytica (HMI-IMSS) were studied. The trophozoites transported adenosine and its analog tubercidin (1 microM) at a significant rate but poor transportation was observed in case of uridine (about 10% relative rate), inosine (3%), thymidine (2%) and formycin B (1%). The Km for adenosine was 160 +/- 42 microM. Unlabeled nucleosides (100 microM) inhibited adenosine and tubercidin transport. Adenosine related compounds 5'-deoxyadenosine and nebularin inhibited adenosine and tubercidine transport by 50% or more. However, inosine related compounds guanosine, 3'-deoxyinosine and formycin B were less inhibitory. The pyrimidine nucleosides uridine, thymidine and cytidine were poorly inhibitory. 6-[(4 nitrobenzyl)-mercapto] purine ribonucleoside, an inhibitor of mammalian nucleoside transporter, inhibited adenosine or tubercidin transport in E. histolytica variably between 0-30% at 10 microM, but dilazep, a known inhibitor, was inactive upto 10 microM. Increase in temperature from 22 degrees C to 33 degrees C enhanced the rate of transport of adenosine 4.5 fold, tubercidin 7.3 fold and of inosine 4 fold. These findings along with the structure activity figures suggested that transport was mediated and not passive.     

Tumor collagenase stimulatory factor (TCSF) expression and localization in human lung and breast cancers

Evidence from in vitro studies indicates that increased proliferation of epithelial cells and secretion of fluid by these cells may be important factors in the progressive enlargement of renal cysts. The rate of cellular proliferation and fluid secretion by cyst epithelium in vitro can be strikingly accelerated by cyclic adenosine 3'5' monophosphate (cAMP) and agonists that lead to the production of this nucleotide. The extent to which renal cAMP content is increased in polycystic kidneys is unknown. In the current study, we determined the amount of this nucleotide in intact kidneys, cyst fluid, plasma, and urine in nonazotemic mice (DBA/2FG-pcy/pcy) with a slowly progressive form of inherited polycystic kidney disease (PKD). In 45 pcy/pcy mice studied 20, 45, or 70 days after birth, the total kidney cAMP content was 0.22 +/- 0.01, 0.46 +/- 0.02, and 0.90 +/- 0.05 pmol/mg tissue, respectively. By contrast, in 37 control DBA/2J mice the levels of cAMP at identical times remained relatively constant at 0.22 +/- 0.01, 0.21 +/- 0.01, and 0.29 +/- 0.01 pmol/mg tissue, respectively. In 70-day-old nonazotemic pcy/pcy mice with normal serum levels of parathyroid hormone, cAMP generated by the kidneys (nephrogenous cAMP) was 22.9 +/- 2.8 nmol/100 mL creatinine clearance, compared with 6.5 +/- 1.3 in normal animals of the same age (P < 0.001). The cyst fluids of 70-day-old pcy/pcy mice contained a lipid that increased transepithelial secretion of fluid by MDCK monolayers from a baseline of 0.012 +/- 0.002 to 0.136 +/- 0.008 microL/cm2/hr (P < 0.05). This lipid also stimulated cellular proliferation by monolayers of cultured MDCK and LLC-PK1 cells 2.5- and 7.9-fold (P < .05), respectively, and stimulated cAMP accumulation by these cells 1.6- and 2.0-fold (P < .05), respectively. These studies illustrate that renal cAMP production and excretion increase in concert with the cystic enlargement of the kidneys in DBA/2FG-pcy/pcy mice and identify a lipid cAMP agonist in murine renal cystic kidney disease.