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(1/1150) Five caffeine metabolite ratios to measure tobacco-induced CYP1A2 activity and their relationships with urinary mutagenicity and urine flow.

To choose a sensitive protocol to discriminate populations exposed and not exposed to inducers, five urinary metabolite ratios (MRs) [MR1 (17X + 17U)/137X, MR2 (5-acetylamino-6-formylamino-3-methyluracil [AFMU] + 1X + 1U)/17U, MR3 (17X/137X), MR4 (AFMU + 1X + 1U + 17X + 17U)/137X, and MR5 (AFMU + 1X + 1U)/17X] were calculated in 4-5 h and 0-24 h urine samples after caffeine intake. One hundred twenty-five healthy volunteers (59 nonsmokers and 66 smokers) were included in the study. All ratios showed a log-normal distribution. MR2 in the two time intervals was the only ratio nondependent on the urine flow. Differences between nonsmokers and smokers could be detected with all ratios at 4-5 h. However, only MR2 and, to a lesser extent, MR5 allowed the discrimination of higher cytochrome P450 1A2 (CYP1A2) activity in smokers in the 0-24 h sample. Although smokers had increased urinary mutagenicity in relation to nonsmokers, a significant association between MRs and urine mutagenicity was observed only with MR2 in the 4-5 h interval; this ratio/time schedule being that of higher association with tobacco consumption. The most flow-dependent ratios, MR1, MR3, and MR4, were closely correlated with each other at the two intervals. The flow dependency profile of each ratio may explain their different power to indicate both tobacco exposure and tobacco-derived mutagenicity. In conclusion, MR2 in the period of 4-5 h after caffeine intake seems preferable, especially at high urine flow rates.  (+info)

(2/1150) Maintenance of adenosine A1 receptor function during long-term anoxia in the turtle brain.

It has been established that adenosine has a critical role in the extraordinary ability of the turtle brain to survive anoxia. To further investigate this phenomenon we compared rat and turtle brain adenosine A1 receptors using cyclopentyl-1,3-dipropylxanthine, 8-[dipropyl-2,3-3H(N)] ([3H]DPCPX) saturation binding analyses and determined the effects of prolonged anoxia (6, 12, and 24 h) on the adenosine A1 receptor of the turtle brain. The rat brain had a 10-fold greater density of A1 receptors compared with the turtle [rat cortex receptor density (Bmax) = 1,400 +/- 134.6 fmol/mg protein, turtle forebrain Bmax = 103.2 +/- 4.60 fmol/mg protein] and a higher affinity [dissociation constant (Kd) rat cortex = 0.328 +/- 0.035 nM, Kd turtle forebrain = 1.16 +/- 0.06 nM]. However, the turtle Kd is within the reported mammalian range, and the Bmax is similar to that reported for other poikilotherms. Unlike the mammal, in which A1 receptor function is rapidly compromised in anoxia, in the turtle forebrain no significant changes in the A1 receptor population were seen during 24-h anoxia. However, in the hindbrain, whereas the Bmax remained unchanged, the Kd significantly decreased from 2.1 to 0.5 nM after 6 h anoxia and this higher affinity was maintained at 12- and 24-h anoxia. These findings indicate that, unlike the GABAA receptor, the protective effectiveness of adenosine in the anoxic turtle brain is not related to an enhanced receptor number. Protection from a hypoxia-induced compromise in A1 receptor function and an increased A1 sensitivity in the hindbrain may be important factors for maintaining the adenosine-mediated downregulation of energy demand during long-term anoxia.  (+info)

(3/1150) Metabolism of methionine and biosynthesis of caffeine in the tea plant (Camellia sinensis L.).

1. Caffeine biosynthesis was studied by following the incorporation of 14C into the products of L-[Me-14C]methionine metabolism in tea shoot tips. 2. After administration of a 'pulse' of L-[Me-14C]methionine, almost all of the L-[Me-14C]methionine supplied disappeared within 1 h, and 14C-labelled caffeine synthesis increased throughout the experimental periods, whereas the radioactivities of an unknown compound and theobromine were highest at 3 h after the uptake of L-[Me-14C]methionine, followed by a steady decrease. There was also slight incorporation of the label into 7-methylxanthine, serine, glutamate and aspartate, disappearing by 36 h after the absorption of L-[Me-14C]methionine. 3. The radioactivities of nucleic acids derived from L-[Me-14C]methionine increased rapidly during the first 12 h incubation period and then decreased steadily. Sedimentation analysis of nucleic acids by sucrose-gradient centrifugation showed that methylation of nucleic acids in tea shoot tips occurred mainly in the tRNA fraction. The main product among the methylated bases in tea shoot tips was identified as 1-methyladenine. 4. The results indicated that the purine ring in caffeine is derived from the purine nucleotides in the nucleotide pool rather than in nucleic acids. A metabolic scheme to show the production of caffeine and related methylxanthines from the nucleotides in tea plants is discussed.  (+info)

(4/1150) A2B adenosine receptors mediate relaxation of the pig intravesical ureter: adenosine modulation of non adrenergic non cholinergic excitatory neurotransmission.

1. The present study was designed to characterize the adenosine receptors involved in the relaxation of the pig intravesical ureter, and to investigate the action of adenosine on the non adrenergic non cholinergic (NANC) excitatory ureteral neurotransmission. 2. In U46619 (10(-7) M)-contracted strips treated with the adenosine uptake inhibitor, nitrobenzylthioinosine (NBTI, 10(-6) M), adenosine and related analogues induced relaxations with the following potency order: 5'-N-ethylcarboxamidoadenosine (NECA) = 5'-(N-cyclopropyl)-carboxamidoadenosine (CPCA) = 2-chloroadenosine (2-CA) > adenosine > cyclopentyladenosine (CPA) = N6-(3-iodobenzyl)-adenosine-5'-N-methylcarboxamide (IB-MECA) = 2-[p-(carboxyethyl)-phenylethylamino]-5'-N-ethylcarboxamidoaden os ine (CGS21680). 3. Epithelium removal or incubation with indomethacin (3 x 10(-6) M) and L-N(G)-nitroarginine (L-NOARG, 3 x 10(-5) M), inhibitors of prostanoids and nitric oxide (NO) synthase, respectively, failed to modify the relaxations to adenosine. 4. 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 10(-8) M) and 4-(2-[7-amino-2-(2-furyl) [1,2,4]-triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385, 3 x 10(-8) M and 10(-7) M), A1 and A2A receptor selective antagonists, respectively, did not modify the relaxations to adenosine or NECA. 8-phenyltheophylline (8-PT, 10(-5) M) and DPCPX (10(-6) M), which block A1/A2-receptors, reduced such relaxations. 5. In strips treated with guanethidine (10(-5) M), atropine (10(-7) M), L-NOARG (3 x 10(-5) M) and indomethacin (3 x 10(-6) M), both electrical field stimulation (EFS, 5 Hz) and exogenous ATP (10(-4) M) induced contractions of preparations. 8-PT (10(-5) M) increased both contractions. DPCPX (10(-8) M), NECA (10(-4) M), CPCA, (10(-4) M) and 2-CA (10(-4) M) did not alter the contractions to EFS. 6. The present results suggest that adenosine relaxes the pig intravesical ureter, independently of prostanoids or NO, through activation of A2B-receptors located in the smooth muscle. This relaxation may modulate the ureteral NANC excitatory neurotransmission through a postsynaptic mechanism.  (+info)

(5/1150) Endogenous interstitial adenosine in isolated myenteric neural networks varies inversely with prevailing PO2.

Isolated myenteric ganglion networks were used in a perifusion protocol to characterize the response of interstitial adenosine levels to changes in prevailing PO2. The biological activity of such adenosine was assessed using inhibition of release of substance P (SP) as a functional measure of adenosine activity, and the effect of altered O2 tension on both spontaneous and elevated extracellular K+ concentration-evoked SP release from networks was determined over a range of PO2 values from hypoxic (PO2 = 54 mmHg) to hyperoxic (PO2 = 566 mmHg). Release of SP was found to be sensitive to PO2, and a linear graded relationship was obtained. Perifusion in the additional presence of the adenosine A1-receptor-selective antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) revealed considerable adenosinergic inhibition with an inverse exponential relationship and hyperoxic threshold PO2. Disinhibition of evoked SP release by DPCPX in the absence of TTX was double that observed in its presence, indicating a neural source for some of the adenosine released during hypoxia. A postulated neuroprotective role for adenosine is consistent with the demonstrated relationship between interstitial adenosine and prevailing O2 tension.  (+info)

(6/1150) Natriuretic and diuretic actions of a highly selective adenosine A1 receptor antagonist.

The natriuretic and diuretic action of a highly selective adenosine A1 receptor (A1AdoR) antagonist, 1,3-dipropyl-8-[2-(5,6-epoxy)norbornyl]xanthine (CVT-124), was investigated in anesthetized rats. CVT-124 (0.1 to 1 mg/kg) caused dose-dependent increases in urine flow and fractional and absolute sodium excretion of by six- to 10-fold and, at 0.1 mg/kg, increased the GFR (1.6+/-0.1 to 2.5+/-0.2 ml/min; P<0.01). There were no changes in BP or heart rate. CVT-124 reduced absolute proximal reabsorption (26+/-3 to 20+/-2 nl/min; P<0.05) despite unchanged proximally measured, single-nephron GFR (SNGFR) (42+/-5 to 44+/-4 nl/min; NS) and thereby decreased fractional proximal reabsorption (60+/-3 to 46+/-4%; P<0.05). Despite increasing distal tubular fluid flow rate (5.4+/-0.7 to 9.7+/-0.9 nl/min; P<0.001), it reduced the proximal-distal difference in SNGFR (before: 9.4+/-1.0 versus during CVT-124: 4.6+/-1.5 nl/min; P<0.01), suggesting that it had blunted the effects of the macula densa on SNGFR. Direct measurements of maximal tubuloglomerular feedback (TGF) responses were made from proximal stop flow pressure (PSF) during orthograde loop perfusion from the proximal tubule with artificial tubular fluid at 40 nl/min. TGF was blunted by intravenous CVT-124 (0.5 mg/kg; deltaPSF with vehicle: 8.3+/-0.6 versus CVT-124: 6.5+/-0.3 mm Hg; n = 9; P<0.01). In conclusion, A1AdoR blockade reduces proximal reabsorption and uncouples it from glomerular filtration. It increases distal delivery of fluid yet does not activate a macula densa-dependent fall in SNGFR because it blunts the TGF response. Natriuresis accompanied by blockade of proximal glomerulotubular balance and TGF characterizes a new class of diuretic drugs.  (+info)

(7/1150) Characterization and tissue location of the neural adenosine receptor in the rat ileum.

1. The aim of the present investigation was to characterize and determine the tissue location of the adenosine receptors present in the rat ileum using a method that detects drug action on the cholinergic nerves innervating the longitudinal and circular muscles. 2. The non-selective adenosine agonist, NECA (10 and 100 nM) caused significant concentration-related reductions in the circular muscle responses to transmural stimulation over the frequency range of 2.5-40 Hz, but did not affect the responses of the longitudinal muscle, nor did it reduce the muscle responses of the guinea-pig ileum. 3. The affinity order of antagonists at inhibiting the effect of NECA on the circular muscle was: CPDPX>8-PT>DMPX with apparent pA2 values of 9.31, 7.54 and 5.63 respectively. CPDPX (10-100 nM) caused parallel displacements of the concentration-effect curves to CPA with a pKb value of 9.15 and Schild slope of 1.03. 4. The agonists previously tested in the rat jejunum peristaltic reflex preparation were also shown to inhibit responses of the rat ileum in the following decreasing order of potency: CPA>NECA>2-CADO>R-PIA>S-PIA>>PAA. In addition, CHA and CCPA were also potent agonists. NECA (100 nM) and CPA (32 nM) did not inhibit carbachol (1 microM)-induced tone of tissues pre-treated with TTX (1 microM). 5. In conclusion, the rat ileum contains inhibitory A1 adenosine receptors situated on cholinergic nerve endings innervating the circular muscle.  (+info)

(8/1150) Cytochrome P450 isoform selectivity in human hepatic theobromine metabolism.

AIMS: The plasma clearance of theobromine (TB; 3,7-dimethylxanthine) is known to be induced in cigarette smokers. To determine whether TB may serve as a model substrate for cytochrome P450 (CYP) 1A2, or possibly other isoforms, studies were undertaken to identify the individual human liver microsomal CYP isoforms responsible for the conversion of TB to its primary metabolites. METHODS: The kinetics of formation of the primary TB metabolites 3-methylxanthine (3-MX), 7-methylxanthine (7-MX) and 3,7-dimethyluric acid (3,7-DMU) by human liver microsomes were characterized using a specific hplc procedure. Effects of CYP isoform-selective xenobiotic inhibitor/substrate probes on each pathway were determined and confirmatory studies with recombinant enzymes were performed to define the contribution of individual isoforms to 3-MX, 7-MX and 3,7-DMU formation. RESULTS: The CYP1A2 inhibitor furafylline variably inhibited (0-65%) 7-MX formation, but had no effect on other pathways. Diethyldithiocarbamate and 4-nitrophenol, probes for CYP2E1, inhibited the formation of 3-MX, 7-MX and 3,7-DMU by approximately 55-60%, 35-55% and 85%, respectively. Consistent with the microsomal studies, recombinant CYP1A2 and CYP2E1 exhibited similar apparent Km values for 7-MX formation and CYP2E1 was further shown to have the capacity to convert TB to both 3-MX and 3,7-DMU. CONCLUSIONS: Given the contribution of multiple isoforms to 3-MX and 7-MX formation and the negligible formation of 3,7-DMU in vivo, TB is of little value as a CYP isoform-selective substrate in humans.  (+info)