Doxofylline: A “Novofylline”

Xanthines such as theophylline have been used in the treatment of lung diseases since the early 1900's, but have a major drawback of a very narrow therapeutic window and many drug/drug interactions. With the increasing availability of other classes of drugs, this has limited the use of xanthnes. Doxofylline is a xanthine molecule that appears to be both bronchodilator and anti-inflammatory with an improved therapeutic window over conventional xanthines such as theophylline and the evidence supporting the effects of doxofylline in the treatment of lung diseases is discussed.     

Doxofylline,a novofylline inhibits lung inflammation induced by lipopolysacharide in the mouse

RationalDoxofylline is a xanthine drug that has been used as a treatment for respiratory diseases for more than 30 years. In addition to doxofylline being a bronchodilator, some studies have indicated that doxofylline also has anti-inflammatory properties, although little is known about the effect of this drug on lung inflammation.ObjectivesWe have investigated the actions of doxofylline against the effects of Escherichia coli LPS in the lungs of BALB/c mice.MethodsAnimals have been treated with doxofylline (0.1, 0.3 and 1 mg/kg i.p.) 24, -and 1 h before, and 6 h after intra-nasal instillation of LPS (10 μg/mouse). Readouts were performed 24 h later.ResultsDoxofylline at 1 and 0.3, but not at 0.1 mg/kg, significantly inhibit neutrophil recruitment to the lung induced by LPS (LPS: 208.4 ± 14.5 versus doxofylline: 1 mg/kg: 106.2 ± 4.8; 0.3 mg/kg: 105.3 ± 10.7 × 10⁴ cells/ml). Doxofylline significantly inhibited IL-6 and TNF-α release into BAL fluid in comparison to LPS-treated animals (LPS: 1255.6 ± 143.9 versus doxofylline 1 mg/kg: 527.7 ± 182.9; 0.3 mg/kg: 823.2 ± 102.3 pg/ml). Intra-vital microscopy of the tracheal tissue demonstrated that doxofylline significantly reduced LPS-mediated leukocyte adhesion to the vessel wall (LPS: 5.9 ± 2.4 versus doxofylline 0.3 mg/kg: 1.78 ± 0.87 cells/100 μm/15 s). Similarly, intra-vital microscopy of cremaster muscle demonstrated that doxofylline significantly reduced LPS-mediated leukocyte transmigration across the blood vessel wall (LPS: 9.3 ± 3.5 versus doxofylline0.3 mg/kg: 3.1 ± 1.87 cells/100 μm²). Doxofylline (0.1–10 μM) also reduced fMLP-induced leukocyte migration in vitro, achieving a maximum effect at 10 μM (fMLP: 37.8 ± 0.5 versus doxofylline 10 μM: 15.1 ± 1.2 cells × 10⁴/ml).ConclusionDoxofylline inhibits LPS-induced inflammation in the lungs of mice.     

多索茶碱对慢性阻塞性肺疾病患者血清细胞因子的影响

目的探讨多索茶碱对慢性阻塞性肺疾病（COPD）患者血清IL-8、TNF—α、IL-10水平的影响,评价多索茶碱对COPD患者的疗效。方法AECOPD患者随机分为两组,治疗组采用多索茶碱治疗,对照组采用氨茶碱治疗,治疗前后观察血清细胞因子（IL-8、TNF—α、IL-10）、肺功能、血气分析的变化。结果两组治疗后均能改善患者肺功能及血气分析指标,改善血清IL-8、TNF-α、IL-10的表达,且治疗组优于对照组。结论多索茶碱能明显改善COPD患者肺功能及血气分析指标,对COPD患者有良好的疗效,值得临床广泛应用。     

Tracheal relaxation from combinations of xanthines and of aβ 2-receptor agonist and xanthines

The present study examined relaxant effects of combinations of different bronchodilator drugs in guinea pig isolated carbachol-contracted tracheal preparations. Theβ ₂-receptor agonist terbutaline (10 and 30 ng/ml), the adenosine receptor antagonist theophylline (10, 20, and 40μg/ml), and the adenosine nonblocking xanthine, enprofylline (2.5, 5, and 10μg/ml) each produced concentration-dependent threshold (up to 25% of maximum) relaxant responses. Combinations of theophylline and enprofylline produced effects that were almost identical to the calculated algebraic sum of effects of the individual drugs. Thus significant additive (P < 0.001–0.05), but no overadditive (P > 0.5) interactions occurred between the 2 xanthines. However, with terbutaline either xanthine produced more than additive relaxant effects. The most pronounced interaction was between terbutaline 30 ng/ml and enprofylline 10μg/ml, which produced 61% relaxation compared to the calculated 33.5% (P < 0.01). It is concluded that airway smooth muscle relaxant effects of combinations of enprofylline and theophylline are additive but when either of these 2 xanthines is combined with aβ ₂-adrenoceptor stimulant more than additive airway relaxation may occur. This interaction is compatible with different cellular mechanisms of action of xanthines andβ-receptor agonists. It cannot be excluded that phosphodiesterase inhibition by xanthines has contributed to the overadditive interaction with theβ ₂-receptor agonist.     

Potentiation of the thermogenic antiobesity effects of ephedrine by dietary methylxanthines: adenosine antagonism or phosphodiesterase inhibition?

Current concepts about the mechanisms underlying the therapeutic effects of dietary methylxanthines (caffeine, theophylline, and theobromine) favor their actions as antagonists of adenosine receptors, and attribute their other possible modes of action, namely those associated with translocation of intracellular calcium, inhibition of phosphodiesterase enzyme (PDE) activity, or the release of catecholamines, to high (near-toxic) doses. From studies measuring the respiration rate of brown adipose tissue (BAT), evidence is provided here that at concentrations compatible with therapeutic doses, the ability of methylxanthines (25 to 50 mumol/L) to potentiate the thermogenic effect of the sympathomimetic drug, ephedrine (0.25 mumol/L), particularly under conditions of caloric restriction, involves a minor contribution of adenosine antagonism, but could mainly be explained by the inhibition of PDE activity. In view of current interest in the pharmacological stimulation of metabolic rate to assist the management of obesity with low-calorie regimens, the targeting of PDE activity is therefore a rational approach in the search for drugs that could potentiate sympathomimetic stimulation of metabolic rate.     

Elevated cyclic AMP and PDE4 inhibition induce chemokine expression in human monocyte-derived macrophages

Macrophages are central mediators of the innate immune system that can be differentiated from monocytes upon exposure to cytokines. While increased cyclic adenosine monophosphate (cAMP) levels are known to inhibit many lipopolysaccharide-elicited macrophage inflammatory responses, the effects of elevated cAMP on monocyte/macrophage differentiation are not as well understood. We show here that during differentiation, cAMP agonists can cause a large increase in the mRNA and protein levels of several of the pro-inflammatory CXCL and CCL chemokines. The cAMP mediator-exchange protein activated by cAMP (Epac) contributes substantially to the increase in these chemokines. These chemokines are known to play an important role in the regulation of immune responses, particularly regarding the pathogenesis of asthma and chronic obstructive pulmonary disorder. We also found that a selective cAMP-degrading phosphodiesterase (PDE) 4 inhibitor can potentiate the chemokine expression elicited by low-dose forskolin or Prostaglandin E2 (PGE₂). These data suggest that chemokine receptor antagonists administered in conjunction with a PDE4 inhibitor may improve both the efficacy and safety of PDE4-inhibitor therapy for chronic inflammatory disorders.     

CVT‐510: A Selective A1 Adenosine Receptor Agonist

Adenosine is an endogenous nucleoside that has potent antiarrhythmic effects on paroxysmal supraventricular tachycardia (PSVT) due to its negative dromotropic effects on the atrioventricular node. In addition to its electrophysiologic effects, adenosine has important effects on vascular smooth muscle cells, inflammatory cells, the central nervous system, and the kidney. Four known adenosine receptor subtypes (A₁, A_2A, A_2B, and A₃) mediate the pleiotropic effects of adenosine in humans. These receptors are coupled to a wide range of second messenger cascades. Activation of the A¹ adenosine receptor accounts for the negative chronotropic and dromotropic effects of adenosine, whereas A_2A, A_2B and A₃ adenosine receptor activation are responsible for such effects as coronary vasodilation, bronchospasm, inhibition of platelet aggregation, and neuronal stimulation. Elucidation of the specific properties of each of the adenosine receptor subtypes has led to the development of selective ligands as potential therapeutic agents. CVT‐510, N‐(3(R)‐tetrahydrofuranyl)‐6‐aminopurine riboside, was developed as a selective A¹ adenosine receptor agonist that specifically targets the atrioventricular node for termination of PSVT. Preliminary clinical trials have shown that CVT‐510 is effective in terminating PSVT and eliminating many of the undesirable adverse effects of adenosine. CVT‐510 is also being explored as a potential agent for controlling the ventricular rate of atrial fibrillation and flutter.     

Theophylline and PDE4 inhibitors in asthma

Over the past three decades, beta -adrenoceptor agonists and glucocorticosteroids have formed the mainstay of treatment for patients with asthma; during this time, only one new drug class, leukotriene receptor antagonists, have been introduced. Theophylline has also been used in the treatment of patients with asthma, although there is a perception that this drug does not offer the patient any advantages over conventional therapeutic strategies. However, a number of clinical studies have documented the efficacy of this orally active drug. The mechanism by which theophylline exerts its well recognized antiinflammatory activity remains to be established but, if explained, could lead to newer drug development with greater efficacy. The development of phosphodiesterase (PDE)4 inhibitors is one such approach, and recent studies have demonstrated the potential utility of this new drug class for the treatment of patients with asthma.(2)     

The effect of selective phosphodiesterase isoenzyme inhibition on neutrophil function in vitro

Neutrophil-derived proteases such as neutrophil elastase (NE) and matrix metalloproteinase (MMP) are implicated in the pathogenesis of Chronic Obstructive Pulmonary Disease (COPD). In this study, the effects of selective phosphodiesterase (PDE) inhibition on NE and MMP-9 release, as well as Myeloperoxidase (MPO) activity and integrin-mediated neutrophil adhesion to human umbilical vein endothelial cells (HUVECs), were investigated. Human neutrophils were treated with PDE inhibitors (10(-11)-10(-4)M) in the absence and presence of TNF-alpha (tumour necrosis factor) (100 U ml(-1)) for 30 min, prior to fMLP activation. After 45 min, the cells were removed and NE, MPO and MMP-9 release assessed. In the adhesion studies, the neutrophils were radio-labelled with 51Cr, stimulated and immediately transferred to cultured HUVEC monolayers for 30 min, prior to assessment of adhesion. TNF-alpha (100 U ml(-1)) acted synergistically with fMLP in stimulating azurophil degranulation with respect to both MPO activity (P<0.01) and NE release (P<0.01). In contrast, an additive effect was observed with TNF-alpha and fMLP with regard to MMP-9 release and neutrophil adhesion to HUVECs. The PDE4 inhibitors, roflumilast, roflumilast N-oxide, cilomilast and rolipram significantly suppressed MPO, NE and MMP-9 release in both the presence and absence of TNF-alpha (P<0.05; n=6-10) and also reduced neutrophil adhesion to HUVECs. In contrast, milrinone, a PDE3 inhibitor and the non-selective PDE inhibitor, theophylline did not inhibit azurophil degranulation under any of the experimental conditions. These data provide further evidence that selective PDE4 isoenzyme inhibitors can inhibit neutrophil degranulation, effects not shared by PDE3 inhibitors or theophylline.     

EVIDENCE FOR A ROLE OF CYCLIC AMP IN NEUROMUSCULAR TRANSMISSION

Experiments were undertaken to determine if the effects of epinephrine in promoting neuromuscular transmission were mediated by adenosine 3':5'-cyclic phosphate (cyclic AMP). Dibutyryl cyclic AMP and the methyl xanthines, theophylline and caffeine (which inhibit cyclic AMP hydrolysis), were found to increase the amplitude of the end plate potential in the isolated rat diaphragm. Like epinephrine (which is known to promote cyclic AMP synthesis), these agents appear to facilitate the release of acetylcholine from the motor neuron. This interpretation is supported by the observation that theophylline and dibutyryl cyclic AMP markedly increased the frequency but not the amplitude of the spontaneous miniature end plate potentials. In addition, these drugs increased the number of transmitter packets released in response to nerve stimulation. These results are consistent with the view that cyclic AMP plays a role in the release of acetylcholine and in the "defatiguing effect" of epinephrine.     

Effect of adenosine and adenosine analogs on [14C]aminopyrine accumulation by rabbit parietal cells

Adenosine receptors that modulate adenylate cyclase activity have been identified recently in a number of tissues. Adenosine A₂ receptor is stimulatory to adenylate cyclase, whereas adenosine A₁ receptor is inhibitory to adenylate cyclase. We investigated the effect of adenosine and its analogs on [¹⁴C]aminopyrine accumulation by rabbit parietal cells. Rabbit gastric mucosal cells were isolated by enzyme digestion. Parietal cells were enriched by nonlinear percoll gradients. [¹⁴C]Aminopyrine accumulation was used as an indicator of acid secretion. The effect of 2-chloroadenosine on histamine-stimulated [¹⁴C]aminopyrine accumulation was studied. The effects ofN-ethylcarboxamideadenosine, 2-chloroadenosine, stable analogs of adenosine, and adenosine on [¹⁴C]aminopyrine accumulation were assessed. Cyclic AMP content of parietal cells was determined by radioimmunoassay. Histamine and carbachol, known secretagogues, stimulated [¹⁴C]aminopyrine accumulation. 2-Chloroadenosine did not suppress histaminestimulated [¹⁴C]aminopyrine accumulation. 2-Chloroadenosine,N-ethylcarboxamideadenosine, and adenosine dose dependently increased [¹⁴C]aminopyrine accumulation. The order of potency wasN-ethylcarboxamideadenosine > 2-chloroadenosine > adenosine. 8-Phenyltheophylline and theophylline, adenosine-receptor antagonists, or cimetidine did not have significant effects on the increase of AP uptake induced by 2-chloroadenosine. Coadministration of dipyridamole, an adenosine uptake inhibitor, augmented the effect of adenosine on [¹⁴C]aminopyrine accumulation. 2-Chloroadenosine,N-ethylcarboxamideadenosine, and adenosine each induced a significant increase in cellular cyclic AMP. We conclude that there may be adenosine A₂ receptors on rabbit parietal cells which modulate gastric acid secretion.     

Inhibition of tracheal smooth muscle cell proliferation by phosphodiesterase inhibitors

Agents that increase intracellular cyclic 3',5'-adenosine monophosphate (cAMP), such as forskolin, prostaglandin (PG)E₂, salbutamol and 8-bromo-cAMP, have been shownto inhibit the proliferation of airway smooth-muscle (ASM) cells in vitro. However, it has not yet been determined whether selective inhibitors of phosphodiesterase (PDE) isoenzymes III and IV that catalyze cAMPto 5'-adenosine monophosphate have the ability to inhibit ASM cell proliferation. To evaluate the effectsof PDE inhibitors on ASM cell proliferation, ASM cells isolated from bovine tracheae were cultured in the presence of fetal bovine serum (FBS), with or without a non-selective PDE inhibitor (theophylline), a selective PDE III inhibitor (cilostazol), and a selective PDE IV inhibitor (rolipram). The number of ASM cells cultured with 5% FBS was significantly reduced by the presence of theophylline at 10^− 3 and 3 × 10^− 4 mol/L, cilostazol at 10^− 5, 10^− 6 and 10^− 7 mol/L, and rolipram at 10^− 4 and 10^− 5 mol/L. The release of lactic dehydrogenase from ASM cells cultured with any concentration of these agents was not significantly different from that with medium alone. Inhibitors of PDE III and IV were demonstrated to have an inhibitory effect on ASM cell proliferation induced by FBS. Our results suggest the value of the further development of PDE inhibitors for the treatment of hyperplasia of ASM cells characteristic of airway remodeling, in addition to bronchospasm and airway inflammation, in bronchial asthma.     

Adenosine A3 Receptor: A Promising Therapeutic Target in Cardiovascular Disease

Cardiovascular complications are one of the major factors for early mortality in the present worldwide scenario and have become a major challenge in both developing and developed nations. It has thus become of immense importance to look for different therapeutic possibilities and treatments for the growing burden of cardiovascular diseases. Recent advancements in research have opened various means for better understanding of the complication and treatment of the disease. Adenosine receptors have become tool of choice in understanding the signaling mechanism which might lead to the cardiovascular complications. Adenosine A3 receptor is one of the important receptor which is extensively studied as a therapeutic target in cardiovascular disorder. Recent studies have shown that A₃AR is involved in the amelioration of cardiovascular complications by altering the expression of A₃AR. This review focuses towards the therapeutic potential of A₃AR involved in cardiovascular disease and it might help in better understanding of mechanism by which this receptor may prove useful in improving the complications arising due to various cardiovascular diseases. Understanding of A₃AR signaling may also help to develop newer agonists and antagonists which might be prove helpful in the treatment of cardiovascular disorder.     

Adenosine A2A receptor agonists with potent antiplatelet activity

Selected adenosine A_2A receptor agonists (PSB-15826, PSB-12404, and PSB-16301) have been evaluated as new antiplatelet agents. In addition, radioligand-binding studies and receptor-docking experiments were performed in order to explain their differential biological effects on a molecular level. Among the tested adenosine derivatives, PSB-15826 was the most potent compound to inhibit platelet aggregation (EC₅₀ 0.32 ± 0.05 µmol/L) and platelet P-selectin cell-surface localization (EC₅₀ 0.062 ± 0.2 µmol/L), and to increase intraplatelets cAMP levels (EC₅₀ 0.24 ± 0.01 µmol/L). The compound was more active than CGS21680 (EC₅₀ 0.97±0.07 µmol/L) and equipotent to NECA (EC₅₀ 0.31 ± 0.05 µmol/L) in platelet aggregation induced by ADP. In contrast to the results from cAMP assays, K_i values determined in radioligand-binding studies were not predictive of the A_2A agonists’ antiplatelet activity. Docking studies revealed the key molecular determinants of this new family of adenosine A_2A receptor agonists: differences in activities are related to π-stacking interactions between the ligands and the residue His264 in the extracellular loop of the adenosine A_2A receptor which may result in increased residence times. In conclusion, these results provide an improved understanding of the requirements of antiplatelet adenosine A_2A receptor agonists.     

Myocardial Phosphodiesterases and Regulation of Cardiac Contractility in Health and Cardiac Disease

Phosphodiesterase (PDE) inhibitors are potent cardiotonic agents used for parenteral inotropic support in heart failure. Contractile effects of these agents are mediated through cAMP-protein kinase A-induced stimulation of I _Ca2+ which ultimately results in increased Ca²⁺-induced sarcoplasmic reticulum Ca²⁺ release. A number of additional effects such as increases in sarcoplasmic reticulum Ca²⁺ stores, stimulation of reverse mode Na⁺–Ca²⁺ exchange, direct or cAMP-mediated effects on sarcoplasmic reticulum ryanodine receptor, stimulation of the voltage-sensitive sarcoplasmic reticulum Ca²⁺ release mechanism, as well as A₁ adenosine receptor blockade could contribute to positive inotropic responses to PDE inhibitors. Moreover, some PDE inhibitors exhibit Ca²⁺ sensitizer properties as they could increase the affinity of troponin C Ca²⁺-binding sites as well as reduce Ca²⁺ threshold for thin myofilament sliding and facilitate cross-bridge cycling. Inotropic responses to PDE inhibitors are significantly reduced in cardiac disease, an effect largely attributed to downregulation of cAMP-mediated signalling due to sustained sympathetic activation. Four PDE isoenzymes (PDE1, PDE2, PDE3 and PDE4) are present in myocardial tissue of various mammalian species, of which PDE3 and PDE4 are particularly involved in regulation of cardiac myocyte contraction. PDE cAMP-hydrolysing activity is preserved in compensated cardiac hypertrophy but significantly reduced in animal models of heart failure. However, clinical studies have not revealed any changes in distribution profile as well as kinetic and regulatory properties of myocardial PDEs in failing human hearts. A reduction of PDE inhibitors-induced contractile responses in heart failure has therefore been ascribed to reduced cAMP synthesis due to uncoupling of adenylyl cyclase from β-adrenoreceptor. In cardiac myocytes, PDEs are targeted to distinct subcellular compartments by scaffolding proteins such as myomegalin, mAKAP and β-arrestins. Over subcellular microdomains, cAMP hydrolysis by PDE3 and PDE4 allows to control the activity of local pools of protein kinase A and therefore the extent of protein kinase A-mediated phosphorylation of cellular proteins.     

Cilostazol (Pletal®): A Dual Inhibitor of Cyclic Nucleotide Phosphodiesterase Type 3 and Adenosine Uptake

Cilostazol (Pletal®), a quinolinone derivative, has been approved in the U.S. for the treatment of symptoms of intermittent claudication (IC) since 1999 and for related indications since 1988 in Japan and other Asian countries. The vasodilatory and antiplatelet actions of cilostazol are due mainly to the inhibition of phosphodiesterase 3 (PDE3) and subsequent elevation of intracellular cAMP levels. Recent preclinical studies have demonstrated that cilostazol also possesses the ability to inhibit adenosine uptake, a property that may distinguish it from other PDE3 inhibitors, such as milrinone. Elevation of interstitial and circulating adenosine levels by cilostazol has been found to potentiate the cAMP‐elevating effect of PDE3 inhibition in platelets and smooth muscle, thereby augmenting anti‐platelet and vasodilatory effects of the drug. In contrast, elevation of interstitial adenosine by cilostazol in the heart has been shown to reduce increases in cAMP caused by the PDE3‐inhibitory action of cilostazol, thus attenuating the cardiotonic effects. Cilostazol has also been reported to inhibit smooth muscle cell proliferation in vitro and has been demonstrated in a clinical study to favorably alter plasma lipids: to decrease triglyceride and to increase HDL‐cholesterol levels. One, or a combination of several of these effects may contribute to the clinical benefits and safety of this drug in IC and other disease conditions secondary to atherosclerosis. In eight double‐blind randomized placebo‐controlled trials, cilostazol significantly increased maximal walking distance, or absolute claudication distance on a treadmill. In addition, cilostazol improved quality of life indices as assessed by patient questionnaire. One large randomized, double‐blinded, placebo‐controlled, multicenter competitor trial demonstrated the superiority of cilostazol over pentoxifylline, the only other drug approved for IC. Cilostazol has been generally well‐tolerated, with the most common adverse events being headache, diarrhea, abnormal stools and dizziness. Studies involving off‐label use of cilostazol for prevention of coronary thrombosis/restenosis and stroke recurrence have also recently been reported.     

PDE4 inhibitors in COPD--a more selective approach to treatment

Chronic obstructive pulmonary disease (COPD) is a serious and mounting global public health problem. Although its pathogenesis is incompletely understood, chronic inflammation plays an important part and so new therapies with a novel anti-inflammatory mechanism of action may be of benefit in the treatment of COPD. Cilomilast and roflumilast are potent and selective phosphodiesterase (PDE)4 inhibitors, with an improved therapeutic index compared with the weak, non-selective PDE inhibitor, theophylline. Unlike theophylline, which is limited by poor efficacy and an unfavourable safety and tolerability profile, the selective PDE4 inhibitors are generally well tolerated, with demonstrated efficacy in improving lung function, decreasing the rate of exacerbations and improving quality of life, with proven anti-inflammatory effects in patients with COPD. Theophylline is a difficult drug to use clinically, requiring careful titration and routine plasma monitoring due to the risk of toxic side effects, such as cardiovascular and central nervous system adverse events, with dose adjustments required in many patients, including smokers, the elderly and some patients on concomitant medications. In contrast, the selective PDE4 inhibitors are convenient medications for both patient and physician alike. Hence these agents represent a therapeutic advance in the treatment of COPD, due to their novel mechanism of action and potent anti-inflammatory effects, coupled with a good safety and tolerability profile.     

Blockage of A2A and A3 adenosine receptors decreases the desensitization of human GABAA receptors microtransplanted to Xenopus oocytes

We previously found that the endogenous anticonvulsant adenosine, acting through A_2A and A₃ adenosine receptors (ARs), alters the stability of currents (I_GABA) generated by GABA_A receptors expressed in the epileptic human mesial temporal lobe (MTLE). Here we examined whether ARs alter the stability (desensitization) of I_GABA expressed in focal cortical dysplasia (FCD) and in periglioma epileptic tissues. The experiments were performed with tissues from 23 patients, using voltage-clamp recordings in Xenopus oocytes microinjected with membranes isolated from human MTLE and FCD tissues or using patch-clamp recordings of pyramidal neurons in epileptic tissue slices. On repetitive activation, the epileptic GABA_A receptors revealed instability, manifested by a large I_GABA rundown, which in most of the oocytes (≈70%) was obviously impaired by the new A_2A antagonists ANR82, ANR94, and ANR152. In most MTLE tissue-microtransplanted oocytes, a new A₃ receptor antagonist (ANR235) significantly improved I_GABA stability. Moreover, patch-clamped pyramidal neurons from human neocortical slices of periglioma epileptic tissues exhibited altered I_GABA rundown on ANR94 treatment. Our findings indicate that antagonizing A_2A and A₃ receptors increases the I_GABA stability in different epileptic tissues and suggest that adenosine derivatives may offer therapeutic opportunities in various forms of human epilepsy.     

Transgenic A1 adenosine receptor overexpression increases myocardial resistance to ischemia

Activation of myocardial A₁ adenosine receptors (A₁AR) protects the heart from ischemic injury. In this study transgenic mice were created using the cardiac-specific α-myosin heavy chain promoter and rat A₁AR cDNA. Heart membranes from two transgene positive lines displayed ≈1,000-fold overexpression of A₁AR (6,574 ± 965 and 10,691 ± 1,002 fmol per mg of protein vs. 8 ± 5 fmol per mg of protein in control hearts). Compared with control hearts, transgenic Langendorff-perfused hearts had a significantly lower intrinsic heart rate (248 beats per min vs. 318 beats per min, P < 0.05), lower developed tension (1.2 g vs. 1.6 g, P < 0.05), and similar coronary resistance. The difference in developed tension was eliminated by pacing. Injury of control hearts during global ischemia, indexed by time-to-ischemic contracture, was accelerated by blocking adenosine receptors with 50 μM 8-(p-sulfophenyl) theophylline but was unaffected by addition of 20 nM N⁶-cyclopentyladenosine, an A₁AR agonist. Thus A₁ARs in ischemic myocardium are presumably saturated by endogenous adenosine. Overexpressing myocardial A₁ARs increased time-to-ischemic contracture and improved functional recovery during reperfusion. The data indicate that A₁AR activation by endogenous adenosine affords protection during ischemia, but that the response is limited by A₁AR number in murine myocardium. Overexpression of A₁AR affords additional protection. These data support the concept that genetic manipulation of A₁AR expression may improve myocardial tolerance to ischemia.     

dl-3-n-butylphthalide inhibits platelet activation via inhibition of cPLA2-mediated TXA2 synthesis and phosphodiesterase

Aberrant platelet activation plays a critical role in the pathogenesis of heart attack and stroke. dl-3-n-butylphthalide (NBP) has been approved in China to treat stroke with multiple mechanisms. The anti-stroke effects of NBP may be related to its antiplatelet effects reported in rats in addition to its antioxidative, antiapoptotic, and angiogenic effects. However, the effects and the underlying mechanisms of NBP on human platelets are not yet clear. In this study, we found that NBP concentration-dependently inhibited human platelet aggregation and ATP release induced by ADP, thrombin, U46619, arachidonic acid, or collagen. NBP also inhibited PAC-1 binding induced by ADP or thrombin and platelet spreading on immobilized fibrinogen. NBP reduced TXA₂ synthesis induced by thrombin or collagen via inhibiting cPLA2 phosphorylation, concomitantly with a marked decrease in intracellular calcium mobilization. Moreover, NBP also inhibited human platelet phosphodiesterase (PDE) and elevated 3,5-cyclic adenosine monophosphate level in platelets. In conclusion, NBP significantly inhibits human platelet activation via inhibition of cPLA2-mediated TXA₂ synthesis and PDE, and may be effective as an antiplatelet drug to treat other arterial thrombotic diseases.