Adenosine 5'-monophosphate induces hypothermia and alters gene expressions in the brain and liver of chicks.

The adenosine A1 receptor is important for body temperature regulation in mammals; however, little is known about its function in avian species. In this study, we investigated the effects of the adenosine A1 receptor agonist and antagonist (adenosine 5'-monophosphate [5'-AMP] and 8 p-sulfophenyl theophylline [8-SPT], respectively) on thermoregulation in chickens. Male chicks were used in this study. After administration of 5'-AMP and 8-SPT, the rectal temperature, plasma metabolites, and gene expressions in the hypothalamus and liver were measured. The rectal temperature was reduced by peripheral administration of 5'-AMP, and the hypothermic effect of 5'-AMP was attenuated by central injection of 8-SPT in chicks. In the hypothalamus, the mRNA level of the agouti-related protein (AgRP) was increased by 5'-AMP administration, whereas it was suppressed by 8-SPT. The plasma levels of free fatty acid were elevated in 5'-AMP-treated chicks and that elevation was suppressed by the 8-SPT treatment. The gene expression of proopiomelanocortin in the hypothalamus was affected by 8-SPT. Nevertheless, the gene expressions of the thermoregulation-related genes, such as the thyrotropin-releasing hormone, were not affected by 5'-AMP and 8-SPT. Hepatic gene expressions related to lipid intake and metabolism were suppressed by 5'-AMP. However, the gene expression of the uncoupling protein was upregulated by 5'-AMP. Based on these results, birds, like mammals, will undergo adenosine A1 receptor-induced hypothermia. In conclusion, it is suggested that 5'-AMP-mediated hypothermia via the adenosine A1 receptor may affect the central melanocortin system and suppress hepatic lipid metabolism in chickens.

The study of long term curative effect of chronic obstructive pulmonary disease in remission stage treated with TCM.

In this study of long term curative effect of chronic obstructive pulmonary disease in remission stage treated with TCM, we have selected 79 patients from January 2013 to January 2015 in our hospital with chronic obstructive pulmonary disease as the research object, we have divided into observation group (40 cases) and control group (39 cases) randomly, the control group received routine treatment, observation group received TCM pulmonary rehabilitation therapy, compare pulmonary function and clinical curative effect of 2 groups of patients, and dyspnea index (Brog index), blood oxygen saturation after 6 and 12 months' treatment. The lung function of the observation group was better than that of control group, the difference was significant (P<0.05). The effective rate of observation group was 97.50%, which was better than that of control group (84.62%), the difference was significant (P<0.05). Brog score, blood oxygen saturation of 2 groups of patients before treatment was not statistically significant (P>0.05); observation group's Brog scores after 6 and12 months' treatment were (2.96 + 0.87), (1.61 + 0.49), oxygen saturation were 94%, 99%, the control group's Brog scores were (4.65 + 0.54), (2.97 + 0.91), oxygen saturation were 86%, 93%, the observation group's indicators were better than that of control group after treatment, the difference was significant (P<0.05). TCM lung rehabilitation treatment of chronic obstructive pulmonary disease has obvious curative effect, it can improve the function of lung, reduce the occurrence of dyspnea, improve patients' tolerance and have obvious long-term curative effect.

Antinociceptive activity of cyclopeptide alkaloids isolated from Ziziphus oxyphylla Edgew (Rhamnaceae).

The current study was designed to evaluate the antinociceptive profile of five cyclopeptide alkaloids isolated from Ziziphus oxyphylla, including Oxyphylline-B 1, Oxyphylline C 2 Oxyphylline-D 3, Nummularin-C 4, and Nummularin-R 5. The effect was studied in acetic acid induced writhing and formalin induced flinching behavior tests, at 2.5 and 5mg/kg i.p. In the post-acetic acid induced writhing test, the compounds significantly ameliorated abdominal constrictions in a dose dependent manner, with compounds 1 and 5 showing 80.98% and 77.87% protection, respectively. When challenged in the formalin induced test, pretreatment of compounds significantly attenuated painful sensation in both phases. Moreover, compounds 1 and 5 were more effective with 45.32% and 75.32% for 1 and 36.77% and 71.10% protection for 5, in the 1st and 2nd phases respectively. The peripheral analgesia was strongly augmented by the central effects of these compounds. The current finding strongly supports the ethnomedicinal use of this valuable medicinal plant in various painful conditions.

Antimutagenic and antirecombinagenic activities of noni fruit juice in somatic cells of Drosophila melanogaster.

Noni, a Hawaiian name for the fruit of Morinda citrifolia L., is a traditional medicinal plant from Polynesia widely used for the treatment of many diseases including arthritis, diabetes, asthma, hypertension and cancer. Here, a commercial noni juice (TNJ) was evaluated for its protective activities against the lesions induced by mitomycin C (MMC) and doxorrubicin (DXR) using the Somatic Mutation and Recombination Test (SMART) in Drosophila melanogaster. Three-day-old larvae, trans-heterozygous for two genetic markers (mwh and flr3 ), were co-treated with TNJ plus MMC or DXR. We have observed a reduction in genotoxic effects of MMC and DXR caused by the juice. TNJ provoked a marked decrease in all kinds of MMC- and DXR-induced mutant spots, mainly due to its antirecombinagenic activity. The TNJ protective effects were concentration-dependent, indicating a dose-response correlation, that can be attributed to a powerful antioxidant and/or free radical scavenger ability of TNJ.

Effects of andrographolide on the pharmacokinetics of aminophylline and doxofylline in rats.

Andrographolide, which is one of the main pharmaceutical ingredients in traditional Chinese medicine Andrographis paniculata, can clear heat, detoxify human body, cool blood and reduce swelling, etc. Respiratory tract infectious diseases have been treated with the combination of andrographolide and theophyllines clinically. As andrographolide inhibits the CYP1A2 activity in vitro, it potentially interacts with theophyllines that are mainly metabolized by CYP1A2. Therefore, we herein studied the effects of andrographolide on the pharmacokinetics of aminophylline and doxofylline in rats. The blood drug concentrations of aminophylline, doxofylline and its metabolite theophylline were determined by HPLC. The theophylline AUC(0-t) was significantly elevated confronting the combination of andrographolide and aminophylline compared to that of the control group (P<0.05). Meanwhile, when only aminophylline was used, the theophylline clearance rate was significantly higher than those in the case of combination (P<0.05). The pharmacokinetics parameters of doxofylline and its metabolite theophylline in the individual administration group showed no significantly different from that combined with andrographolide. The results suggest that andrographolide and aminophylline should not be simultaneously administered because the former may raise the risks of side effects by inhibiting the clearance of the latter. In contrast, it is more secure to combine doxofylline with andrographolide owing to the almost intact pharmacokinetics.

Cardiovascular protection and antioxidant activity of the extracts from the mycelia of Cordyceps sinensis act partially via adenosine receptors.

Mycelia of cultured Cordyceps sinensis (CS) is one of the most common substitutes for natural CS and was approved for arrhythmia in China. However, the role of CS in ameliorating injury during ischemia-reperfusion (I/R) is still unclear. We examined effects of extracts from CS on I/R and investigated the possible mechanisms. Post-ischemic coronary perfusion pressure, ventricular function, and coronary flow were measured using the Langendorff mouse heart model. Oxidative stress of cardiac homogenates was performed using an ELISA. Our results indicate that CS affords cardioprotection possibly through enhanced adenosine receptor activation. Cardioprotection was demonstrated by reduced post-ischemic diastolic dysfunction and improved recovery of pressure development and coronary flow. Treatment with CS largely abrogates oxidative stress and damage in glucose- or pyruvate-perfused hearts. Importantly, observed reductions in oxidative stress [glutathione disulfide (GSSG)]/[GSSG + glutathione] and [malondialdehyde (MDA)]/[superoxide dismutase + MDA] ratios as well as the resultant damage upon CS treatment correlate with functional markers of post-ischemic myocardial outcome. These effects of CS were partially blocked by 8-ρ-sulfophenyltheophylline, an adenosine receptor antagonist. Our results demonstrate a suppressive role of CS in ischemic contracture. Meanwhile, the results also suggest pre-ischemic adenosine receptor activation may be involved in reducing contracture in hearts pretreated with CS.

Presynaptic inhibition of GABAergic synaptic transmission by adenosine in mouse hypothalamic hypocretin neurons.

Hypocretin neurons in the lateral hypothalamus, a new wakefulness-promoting center, have been recently regarded as an important target involved in endogenous adenosine-regulating sleep homeostasis. The GABAergic synaptic transmissions are the main inhibitory afferents to hypocretin neurons, which play an important role in the regulation of excitability of these neurons. The inhibitory effect of adenosine, a homeostatic sleep-promoting factor, on the excitatory glutamatergic synaptic transmissions in hypocretin neurons has been well documented, whether adenosine also modulates these inhibitory GABAergic synaptic transmissions in these neurons has not been investigated. In this study, the effect of adenosine on inhibitory postsynaptic currents (IPSCs) in hypocretin neurons was examined by using perforated patch-clamp recordings in the acute hypothalamic slices. The findings demonstrated that adenosine suppressed the amplitude of evoked IPSCs in a dose-dependent manner, which was completely abolished by 8-cyclopentyltheophylline (CPT), a selective antagonist of adenosine A1 receptor but not adenosine A2 receptor antagonist 3,7-dimethyl-1-(2-propynyl) xanthine. A presynaptic origin was suggested as following: adenosine increased paired-pulse ratio as well as reduced GABAergic miniature IPSC frequency without affecting the miniature IPSC amplitude. Further findings demonstrated that when the frequency of electrical stimulation was raised to 10 Hz, but not 1 Hz, a time-dependent depression of evoked IPSC amplitude was detected in hypocretin neurons, which could be partially blocked by CPT. However, under a higher frequency at 100 Hz stimulation, CPT had no action on the depressed GABAergic synaptic transmission induced by such tetanic stimulation in these hypocretin neurons. These results suggest that endogenous adenosine generated under certain stronger activities of synaptic transmissions exerts an inhibitory effect on GABAergic synaptic transmission in hypocretin neurons by activation of presynaptic adenosine A1 receptors, which may finely regulate the excitability of these neurons as well as eventually modulate the sleep-wakefulness.

In vitro and in vivo evaluation of multilayered pastilles for chronotherapeutic management of nocturnal asthma.

OBJECTIVE: The present work was undertaken with an objective to design a multilayered dosage form of doxofylline, using pastillation technology, for the chronotherapeutic management of nocturnal asthma. RESEARCH DESIGN & METHODS: Pastilles consisting of the drug, polyethylene glycol and colloidal silicon dioxide, were generated using an in-house laboratory-scale pastillation device. The pastilles were further coated with enteric polymers and a floating layer, using conventional coater. The pastilles were subjected to physicochemical analysis, morphological characterization, in vitro drug release studies and in vivo pharmacokinetic studies in rats. RESULTS: It was observed that colloidal silicon dioxide was instrumental in improving the contact angle of the pastilles. The uncoated pastilles released the drug immediately, while the enteric-coated (10% w/w) pastilles were found to have sufficient acid resistance when the coat is applied with 5% (v/v) triethyl citrate as plasticizer. The in vivo blood serum profile indicated that the pastilles coated with the enteric coat and the additional floating coat were effective in significantly delaying the in vivo drug release required for the chronotherapeutic treatment of nocturnal asthma. CONCLUSION: The present work opens a new alternative to the conventional tablet or capsule dosage form for the development of both immediate-release and modified-release drug delivery systems.

Development of a whole-cell screening system for evaluation of the human CYP1A2-mediated metabolism.

Cytochrome P450 1A2 (CYP1A2) is an important member of cytochrome P450 involved in drug metabolism. In this study, a cell line, Huh7-1A2-I-E, with high expression level of CYP1A2 is established based on Huh7 cells. To achieve this, we constructed a recombinant lentiviral vector, pLenti-1A2-I-E, containing a single promoter encoding CYP1A2 followed by an internal ribosome entry site (IRES) to permit the translation of enhanced green fluorescence protein (EGFP). Such a design has greatly facilitated the selection of stable cell lines because the translations of CYP1A2 and EGFP proteins would be based on a single bi-cistronic mRNA. The Huh7-1A2-I-E cells were evaluated as a cell-based model for identification of CYP1A2 inhibitors and for studies of cytotoxicity resulted from CYP-mediated drug metabolism. Treatment of Huh7-1A2-I-E cells and the Huh7-E control cells with aflatoxin B1 showed that cells with CYP1A2 expression are much more sensitive to aflatoxin B1 and the cellular toxicity of aflatoxin B1 in Huh7-1A2-I-E cells could be prevented by furafylline, a CYP1A2 inhibitor. A collection of approximately 200 drugs were screened using this system and results indicate that for most drugs the metabolism by CYP1A2 is unlikely to have made a major contribution to the in vitro cytotoxicity except for thimerosal and evoxine. Several previously unidentified CYP1A2 inhibitors such as evoxine and berberine were also identified in this study.

Cilostazol protects the heart against ischaemia reperfusion injury in a rabbit model of myocardial infarction: focus on adenosine, nitric oxide and mitochondrial ATP-sensitive potassium channels.

1. The present study examined whether or not cilostazol reduces the myocardial infarct size, and investigated its mechanism in a rabbit model of myocardial infarction. 2. Japanese white rabbits underwent 30 min of coronary occlusion, followed by 48 h of reperfusion. Cilostazol (1 and 5 mg/kg) or vehicle was given intravenously 5 min before ischaemia. 8-p-sulfophenyl theophylline (8SPT; an adenosine receptor blocker, 7.5 mg/kg), Nω-nitro-L-arginine methylester (l-NAME; an NOS inhibitor, 10 mg/kg) or 5-hydroxydecanoic acid sodium salt (5-HD; a mitochondrial ATP-sensitive potassium (KATP) channel blocker, 5 mg/kg) was given intravenously 5 min before cilostazol injection. Infarct size was determined as a percentage of the risk area. 3. The myocardial interstitial levels of adenosine and nitrogen oxide (NOx) during ischaemia and reperfusion, and the intensity of myocardial dihydroethidium staining were determined. 4. Infarct size was significantly reduced in the cilostazol 1 mg/kg (38.4% (2.9%)) and cilostazol 5 mg/kg (30.7% (4.7%)) groups compared with that in the control group (46.5% (4.2%)). The infarct size-reducing effect of cilostazol was completely abolished by 8SPT (46.6% (3.5%)), L-NAME (49.0% (5.5%)), or 5HD (48.5% (5.1%)). 8SPT, L-NAME or 5HD alone did not affect the infarct size. Cilostazol treatment significantly increased myocardial levels of adenosine and NOx during ischaemia, and attenuated the intensity of dihydroethidium staining during reperfusion. 5. These findings show that cilostazol reduces the myocardial infarct size by increasing adenosine and NOx levels, attenuating superoxide production and opening the mitochondrial KATP channels. Cilostazol might provide a new strategy for the treatment of coronary heart disease.

Regulation of proxyphylline's release from silicone rubber matrices by the use of osmotically active excipients and a multi-layer system.

In this work we present results on the modification of the release kinetics of a water-soluble model drug (proxyphylline) from silicone rubber (SR) matrices by either: (i) the incorporation of inorganic salts acting as osmotically active excipients in single-layer matrices, containing a uniform concentration of proxyphylline or (ii) by the use of three-layer matrices with distributed proxyphylline load. In relation to (i) our data show that the incorporation of inorganic salts, of varying water solubilities and in different initial loads, accelerated the release of proxyphylline and helped to the stabilization of its declining release rate. Drug release kinetics is supplemented by measurements of concurrent water uptake, and salt release, kinetics. In addition, in order to further investigate the release mechanisms of the drug and the salts, we studied the diffusion and sorption properties of the depleted polymeric matrix along with the morphology and the mechanical properties of the matrices either in the presence or after the depletion of the solutes. The combined information, derived from these techniques, supports a drug release mechanism occurring through an excessively swollen polymer matrix and accelerated by the formation of microscopic cracks generated by the osmotically active excipients. In relation to (ii) we studied a multi-layer device with proxyphylline-loaded inner layer and drug-free outer layers which practically diminished the initial burst effect and allowed the release of about 60% of the drug at a constant rate.

Effect of diethyldithiocarbamate (DDC) and ticlopidine on CYP1A2 activity and caffeine metabolism: an in vitro comparative study with human cDNA-expressed CYP1A2 and liver microsomes.

The aim of the present study was to test the effect of diethyldithiocarbamate (DDC), which is regarded as a cytochrome P450 (CYP) CYP2A6 and CYP2E1 inhibitor, and ticlopidine, an efficient CYP2B6, CYP2C19 and CYP2D6 inhibitor, on the activity of human CYP1A2 and the metabolism of caffeine (1-N-, 3-N- and 7-N-demethylation, and C-8-hydroxylation). The experiment was carried out in vitro using human cDNA-expressed CYP1A2 (Supersomes) and human pooled liver microsomes. The effects of DDC and ticlopidine were compared to those of furafylline (a strong CYP1A2 inhibitor). A comparative in vitro study provides clear evidence that ticlopidine and DDC, applied at concentrations that inhibit the above-mentioned CYP isoforms, potently (as compared to furafylline) inhibit human CYP1A2 and caffeine metabolism, in particular 1-N- and 3-N-demethylation.

Synthesis and structure-activity relationship studies of theophylline analogs on population responses in the rat hippocampus in vitro.

We synthesized several theophylline analogs and tested the hypothesis that these compounds may be nootropic or cognitive enhancers by examining their effects on evoked population spikes recorded extracellularly in the CA1 region of the rat hippocampus. Whereas the length of the carbon chain on N7 had no effect, different size of the terminal lactam ring strongly influenced neuroactivity. Our results suggest that hexahydroazepin-2-one analogs have potential for further development as cognitive enhancers.

Progress curve analysis of CYP1A2 inhibition: a more informative approach to the assessment of mechanism-based inactivation?

Mechanism-based cytochrome P450 inactivation is defined as a time- and NADPH-dependent inactivation that is not reversible upon extensive dialysis. Current methodologies use dilution approaches to estimate the rate of inactivation and offer limited mechanistic insight and are significantly influenced by experimental conditions. We investigated the potential of progress curve analysis because this experimental design allows investigation of both the reversible (K(iapp)) and irreversible (K(i), K(inact)) components of the reaction mechanism. The human liver microsomal CYP1A2 inactivation kinetics of resveratrol, oltipraz, furafylline, and dihydralazine (Fig. 2) were evaluated. The inactivation results for furafylline (K(i), 0.8 microM; K(inact), 0.16 min(-1)) are within 2-fold to published data (K(i), 1.6 microM; K(inact), 0.19 min(-1)). Resveratrol and dihydralazine results are within a 4.3-fold range of published data, which compares well with ranges of estimates of these parameters across publications (e.g., furafylline has estimates ranging of K(i) from 1.6 to 22.3 microM and K(inact) from 0.19 to 0.87 min(-1)). This range of estimates highlights the potential caveats surrounding the existing methodologies that have been previously discussed in depth. In addition to these inactivation parameters, we have been able to demonstrate a variation in balance of reversible versus irreversible inhibition within these inactivators. Oltipraz and resveratrol have K(iapp) values similar to their K(i), indicating a significant early onset reversible inhibition, whereas furafylline and dihydralazine are dominated by irreversible inactivation. This approach allows a more mechanistic investigation of an inactivator and in the future may improve the prediction of clinical drug-drug interactions.

Identification of human hepatic cytochrome P450 enzymes involved in the metabolism of 8-prenylnaringenin and isoxanthohumol from hops (Humulus lupulus L.).

The female flowers of hops (Humulus lupulus L.) are used in the brewing of beer and are under investigation for use in dietary supplements for the management of menopausal symptoms in women. Hop extracts contain the weakly estrogenic compound isoxanthohumol (IX), proestrogenic xanthohumol, and the potent estrogen 8-prenylnaringenin (8PN). Because IX can be metabolized in the human liver to form 8PN, the specific cytochrome P450 (P450) enzymes responsible for this O-demethylation reaction were identified. In addition, the enzymes that convert IX and 8PN to their most abundant metabolites were identified because these metabolic pathways might also affect the estrogenicity of hop preparations. Specifically, the P450 enzymes that catalyze the oxidation of the prenyl side chains of IX and 8PN into trans- or cis-alcohols were investigated. Human liver microsomes and monoclonal antibodies that inhibit specific P450 enzymes were used in combination with liquid chromatography/mass spectrometry to identify the enzymes responsible for these transformations. CYP2C19 was found to catalyze the formation of both cis- and trans-alcohols of the prenyl side chain of 8PN with K(m) values of 14.8 +/- 3.2 and 16.6 +/- 4.6 microM, respectively. CYP2C8 converted 8PN regioselectively to the trans-alcohol of the prenyl group with a K(m) of 3.7 +/- 0.9 microM. Finally, CYP1A2 was found to catalyze the O-demethylation of IX to generate 8PN, with a K(m) value of 17.8 +/- 3.7 microM. These results suggest that the estrogenicity of hop constituents in vivo will depend in part on metabolic conversion that may show individual variation.

Purine P1 receptor-dependent immunostimulatory effects of antiviral acyclic analogues of adenine and 2,6-diaminopurine.

Acyclic nucleoside phosphonates are widely recognised antivirals. The oral prodrugs of prototype compounds, e.g., 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA; adefovir), and 9-(R)-[2-(phosphonomethoxy)propyl]adenine [(R)-PMPA; tenofovir] were approved by FDA for treatment of hepatitis B (Hepsera), and acquired immunodeficiency syndrome (AIDS) (Viread), respectively. A number of acyclic nucleoside phosphonates possess immunostimulatory activity. The present experiments demonstrate that activation of cytokine and chemokine secretion is mediated by adenosine receptors. Included in the study were 9-(R)-[2-(phosphonomethoxy)propyl]adenine [tenofovir], N(6)-cyclopentyl-(R)-9-[2-(phosphonomethoxy)propyl]-2,6-diaminopurine, N(6)-cyclopropyl-(R)-9-[2-(phosphonomethoxy)propyl]-2,6-diaminopurine, and N(6)-isobutyl-9-[2-(phosphonomethoxy)ethyl]-2,6-diaminopurine. All of them activate secretion of tumor necrosis factor-alpha (TNF-alpha), interleukin-10 (IL-10), "regulated on activation of normal T cell expressed and secreted" (RANTES/CCL5), and macrophage inflammatory protein-1alpha (MIP-1alpha/CCL3) in murine macrophages. With exception of MIP-1alpha, the effects were inhibited by antagonists of adenosine A(1), A(2B), and A(3) receptors (not by adenosine A(2A) receptor antagonist). The adenosine A(1) receptor antagonist inhibited TNF-alpha, IL-10, and RANTES, adenosine A(2B) receptor antagonist inhibited TNF-alpha and RANTES, and adenosine A(3) receptor antagonist inhibited IL-10 and RANTES. The suppression is due to decreased transcription of cytokine mRNA. It may be suggested that acyclic nucleoside phosphonates are nonspecific ligands for purine P(1) receptors.

Relationship between bicarbonate and cyclic nucleotide in the promoting effects on head-to-head agglutination in boar spermatozoa.

AIM: To clarify the relationship between bicarbonate and cAMP in the promoting effects on the sperm agglutination. METHODS: Spermatozoa were collected from mature boars, washed and resuspended in a modified Krebs-Ringer HEPES lacking calcium chloride (mKRH). The sperm suspensions were incubated in a water bath (38.5 degrees C) for 60 min and then the percentage of head-to-head agglutinated spermatozoa was determined. RESULTS: Supplementation of the mKRH with sodium bicarbonate (5-10 mM) significantly raised the percentage of head-to-head agglutinated spermatozoa in the samples. The addition of selective inhibitors for calcium/calmodulin-dependent phosphodiesterases (type 1: 8-methoxymethyl-IBMX and vinpocetine, 25-50 micro M) or for cAMP-specific phosphodiesterases (type 4: Ro20-1724 and rolipram, 25-50 microM) enhanced the effect of bicarbonate on sperm agglutination as highly as did the addition of non-selective inhibitors for phosphodiesterases (IBMX and papaverine, 25-50 microM). A calmodulin antagonist (W-7, 2 microM), that potentially blocks the stimulator of the calcium/calmodulin-dependent phosphodiesterases, significantly enhanced the effect of bicarbonate on sperm agglutination. Moreover, a phosphodiesterase-resistant cAMP analogue (cBiMPS, 0.1 mM) markedly induced agglutination in more spermatozoa (76%) after the incubation without bicarbonate and phosphodiesterase inhibitors than did a less potent cAMP analogue (dibutyryl cAMP, 1 mM) (21%), while three kinds of cGMP analogues (0.1-1 mM) had no effect on sperm agglutination. In addition, a cAMP antagonist (Rp-cAMPS, 1 mM) significantly reduced the sperm agglutination resulting from the actions of bicarbonate and IBMX. On the other hand, the effect of bicarbonate was abolished by a change of incubation temperature from 38.5 degrees C to 25 degrees C. CONCLUSION: These findings demonstrate that the bicarbonate-induced agglutination of boar spermatozoa is controlled via the cAMP-mediated, temperature-dependent signaling cascade. This cascade is suppressed by the action of the phosphodiesterase (at least types 1 and 4).

Complementary role of extracellular ATP and adenosine in ischemic preconditioning in the rat heart.

Although adenosine is an important mediator of ischemic preconditioning (IPC), its relative contribution to IPC remains unknown. Because adenosine is formed through the hydrolysis of ATP, the present study investigated the role of ATP and adenosine in IPC. Isolated and buffer-perfused rat hearts underwent IPC by three cycles of 5-min ischemia and 5-min reperfusion before 25 min of global ischemia. The rate-pressure product (RPP) 30 min after reperfusion was taken as an endpoint of functional protection. Interstitial fluid (ISF) adenine nucleotides and adenosine were measured by cardiac microdialysis techniques. Inhibition of IPC-induced recovery of RPP was partial by the adenosine receptor antagonist 8-(p-sulfophenyl)theophylline (SPT; 100 microM) or by the structurally distinct P2Y purinoceptor antagonists suramin (300 microM) or reactive blue (RB; 10 microM) but was additive when SPT was given with suramin or RB. The P2X antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid tetrasodium (50 microM) had no effect on functional protection. The improved functional recovery was not significantly affected by an ecto-5'-nucleotidase inhibitor, alpha,beta-methylene adenosine diphosphate (AMP-CP; 100 microM), alone but was inhibited by AMP-CP plus SPT, suramin, or RB. ISF ATP and adenosine increased temporarily by 10-fold during IPC. AMP-CP augmented the increase in ISF ATP associated with the decrease in ISF adenosine. There was a reciprocal correlation between the ISF concentration of ATP and adenosine in preconditioned hearts. In addition, there was a significant correlation between ISF adenosine and ATP and the inhibitory potency of SPT and suramin or RB against functional protection conferred by IPC. These results suggest that extracellular ATP and adenosine play a complementary role in IPC through P2Y purinoceptors and adenosine receptors, respectively.

The alkaloid rutaecarpine is a selective inhibitor of cytochrome P450 1A in mouse and human liver microsomes.

Rutaecarpine, evodiamine, and dehydroevodiamine are quinazolinocarboline alkaloids isolated from a traditional Chinese medicine, Evodia rutaecarpa. The in vitro effects of these alkaloids on cytochrome P450 (P450)-catalyzed oxidations were studied using mouse and human liver microsomes. Among these alkaloids, rutaecarpine showed the most potent and selective inhibitory effect on CYP1A-catalyzed 7-methoxyresorufin O-demethylation (MROD) and 7-ethoxyresorufin O-deethylation (EROD) activities in untreated mouse liver microsomes. The IC(50) ratio of EROD to MROD was 6. For MROD activity, rutaecarpine was a noncompetitive inhibitor with a K(i) value of 39 +/- 2 nM. In contrast, rutaecarpine had no effects on benzo[a]pyrene hydroxylation (AHH), aniline hydroxylation, and nifedipine oxidation (NFO) activities. In human liver microsomes, 1 microM rutaecarpine caused 98, 91, and 77% decreases of EROD, MROD, and phenacetin O-deethylation activities, respectively. In contrast, less than 15% inhibition of AHH, tolbutamide hydroxylation, chlorzoxazone hydroxylation, and NFO activities were observed in the presence of 1 microM rutaecarpine. To understand the selectivity of inhibition of CYP1A1 and CYP1A2, inhibitory effects of rutaecarpine were studied using liver microsomes of 3-methylcholanthrene (3-MC)-treated mice and Escherichia coli membrane expressing bicistronic human CYP1A1 and CYP1A2. Similar to the CYP1A2 inhibitor furafylline, rutaecarpine preferentially inhibited MROD more than EROD and had no effect on AHH in 3-MC-treated mouse liver microsomes. For bicistronic human P450s, the IC(50) value of rutaecarpine for EROD activity of CYP1A1 was 15 times higher than the value of CYP1A2. These results indicated that rutaecarpine was a potent inhibitor of CYP1A2 in both mouse and human liver microsomes.

Doxofylline: a new generation xanthine bronchodilator devoid of major cardiovascular adverse effects.

Doxofylline (7-(1,3-dioxalan-2-ylmethyl) theophylline) is a novel xanthine bronchodilator which differs from theophylline in that it contains a dioxalane group in position 7. Similarly to theophylline, its mechanism of action is related to the inhibition of phosphodiesterase activities, but in contrast it appears to have decreased affinities towards adenosine A1 and A2 receptors, which may account for its better safety profile. The bronchodilating activities of doxofylline have been demonstrated in clinical trials involving patients with either bronchial asthma or chronic obstructive pulmonary disease. In contrast to other bronchodilators, experimental and clinical studies have shown that the drug is devoid of direct stimulatory effects. This may be of importance because the arrhythmogenic actions of bronchodilators may have a negative impact on the survival of patients with respiratory diseases.