Functional expression of ionotropic purinergic receptors on mouse taste bud cells

Neurotransmitter receptors on taste bud cells (TBCs) and taste nerve fibres are likely to contribute to taste transduction by mediating the interaction among TBCs and that between TBCs and taste nerve fibres. We investigated the functional expression of P2 receptor subtypes on TBCs of mouse fungiform papillae. Electrophysiological studies showed that 100 μ m ATP applied to their basolateral membranes either depolarized or hyperpolarized a few cells per taste bud. Ca²⁺ imaging showed that similarly applied 1 μ m ATP, 30 μ m BzATP (a P2X₇ agonist), or 1 μ m 2MeSATP (a P2Y₁ and P2Y₁₁ agonist) increased intracellular Ca²⁺ concentration, but 100 μ m UTP (a P2Y₂ and P2Y₄ agonist) and α,β-meATP (a P2X agonist except for P2X₂, P2X₄ and P2X₇) did not. RT-PCR suggested the expression of P2X₂, P2X₄, P2X₇, P2Y₁, P2Y₁₃ and P2Y₁₄ among the seven P2X subtypes and seven P2Y subtypes examined. Immunohistostaining confirmed the expression of P2X₂. The exposure of the basolateral membranes to 3 m m ATP for 30 min caused the uptake of Lucifer Yellow CH in a few TBCs per taste bud. This was antagonized by 100 μ m PPADS (a non-selective P2 blocker) and 1 μ m KN-62 (a P2X₇ blocker). These results showed for the first time the functional expression of P2X₂ and P2X₇ on TBCs. The roles of P2 receptor subtypes in the taste transduction, and the renewal of TBCs, are discussed.     

Slow excitatory synaptic transmission mediated by P2Y1 receptors in the guinea-pig enteric nervous system

Electrophysiological recording was used to study a type of slow excitatory postsynaptic potential (slow EPSP) that was mediated by release of ATP and its action at P2Y₁ receptors on morphologically identified neurones in the submucosal plexus of guinea-pig small intestine. MRS2179, a selective P2Y₁ purinergic receptor antagonist, blocked both the slow EPSP and mimicry of the EPSP by exogenously applied ATP. Increased conductance accounted for the depolarization phase of the EPSP, which occurred exclusively in neurones with S-type electrophysiological behaviour and uniaxonal morphology. The purinergic excitatory input to the submucosal neurones came from neighbouring neurones in the same plexus, from neurones in the myenteric plexus and from sympathetic postganglionic neurones. ATP-mediated EPSPs occurred coincident with fast nicotinic synaptic potentials evoked by the myenteric projections and with noradrenergic IPSPs evoked by sympathetic fibres that innervated the same neurones. The P2Y₁ receptor on the neurones was identified as a metabotropic receptor linked to activation of phospholipase C, synthesis of inositol 1,4,5-trisphosphate and mobilization of Ca²⁺ from intracellular stores.     

Sex-dependent regulation of hypothalamic neuropeptide Y-Y1 receptor gene expression in moderate/high fat, high-energy diet-fed mice

In this study we investigated whether long-term consumption of a moderate/high fat (MHF), high-energy diet can affect the gene expression of the Y₁ receptor (Y₁R) for neuropeptide Y (NPY) in the dorsomedial (DMH), ventromedial (VMH), arcuate (ARC) and paraventricular (PVN) hypothalamic nuclei of male and female Y₁R/LacZ transgenic mice, carrying the murine Y₁R promoter linked to the LacZ gene. MHF diet-fed male mice showed an increased consumption of metabolizable energy that was associated with a significant increase in body weight as compared with chow-fed controls. In parallel, consumption of a MHF diet for 8 weeks significantly decreased Y₁R/LacZ transgene expression in the DMH and VMH of male mice whereas no changes were found in the ARC and PVN. Leptin treatment reduced body weight of both MHF diet- and chow-fed male mice but failed to prevent the decrease in Y₁R/LacZ transgene expression apparent in the DMH and VMH of male mice after 8 weeks of MHF diet intake. Conversely, no significant changes of metabolizable energy intake, body weight or hypothalamic β-galactosidase expression were found in MHF diet-fed female Y₁R/LacZ transgenic mice. A gender-related difference of Y₁R/LacZ transgenic mice was also observed in response to leptin treatment that failed to decrease body weight of both MHF diet- and chow-fed female mice. Results herein demonstrate that Y₁R/LacZ FVB mice show a sexual dimorphism both on energy intake and on nucleus-specific regulation of the NPY Y₁R system in the hypothalamus. Overall, these results provide new insights into the mechanism by which diet composition affects the hypothalamic circuit that controls energy homeostasis.     

ATP participates in three excitatory postsynaptic potentials in the submucous plexus of the guinea pig ileum

Synaptic transmission between neurones intrinsic to the wall of the intestine involves multiple neurotransmitters. This study aimed to identify neurotransmitters responsible for non-cholinergic excitatory synaptic transmission in the submucous plexus of the guinea pig ileum. Intracellular recordings were made from secretomotor and vasodilator neurones. A single electrical stimulus to a fibre tract evoked excitatory postsynaptic potentials (EPSPs) with three different time courses – fast, slow and an EPSP with an intermediate time course (latency 96 ms, duration 1.2 s). In all neurones, blocking nicotinic receptors reduced fast EPSPs, but they were abolished in only 57 of 78 neurones. Fast EPSPs were also reduced by P2 purinoceptor blockade (5 of 27 neurones) or 5-HT₃ receptor blockade (3 of 20 neurones). The intermediate EPSP was abolished by P2 receptor blockade (13 of 13 neurones) or by the specific P2Y₁ receptor antagonist MRS 2179 (5 of 5 neurones) and was always preceded by a nicotinic or mixed nicotinic/purinergic fast EPSP. Intermediate EPSPs were observed in over half of all neurones including most non-cholinergic secretomotor neurones identified by immunoreactivity for vasoactive intestinal peptide. The slow EPSP evoked by a single pulse stimulus was also abolished by P2 receptor blockade (5 of 5 neurones) or by MRS 2179 (3 of 3 neurones). We conclude that fast EPSPs in submucous neurones are mediated by acetylcholine acting at nicotinic receptors, ATP acting at P2X receptors and 5-HT acting at 5-HT₃ receptors. Both the intermediate EPSP and the single stimulus slow EPSP are mediated by ATP acting at P2Y₁ receptors.     

Antiplatelet Effect of Clopidogrel Can Be Reduced by Calcium-Channel Blockers

Purpose

Clopidogrel is metabolized by the hepatic cytochrome P450 (CYP) system into its active thiol metabolite. CYP3A4 is involved in the metabolism of both clopidogrel and dihydropyridine calcium channel blockers (CCBs). A few reports have suggested an inhibitory interaction between CCBs and clopidogrel. Accordingly, the aim of this study was to determine the effect of CCBs on the antiplatelet activity of clopidogrel by serial P2Y12 reaction unit (PRU) measurements.

Materials and Methods

We assessed changes in antiplatelet activity in patients receiving both clopidogrel and CCBs for at least 2 months prior to enrollment in the study. The antiplatelet activity of clopidogrel was measured by VerifyNow P2Y12 assay in the same patient while medicated with CCBs and at 8 weeks after discontinuation of CCBs. After discontinuation of the CCBs, angiotensin receptor blockers were newly administered to the patients or dosed up for control of blood pressure.

Results

Thirty patients finished this study. PRU significantly decreased after discontinuation of CCBs (238.1±74.1 vs. 215.0±69.3; p=0.001). Of the 11 patients with high post-treatment platelet reactivity to clopidogrel (PRU≥275), PRU decreased in nine patients, decreasing below the cut-off value in seven of these nine patients after 8 weeks. Decrease in PRU was not related to CYP2C19 genotype.

Conclusion

CCBs inhibit the antiplatelet activity of clopidogrel.     

Cytochrome P450 2C19 polymorphism is associated with reduced clopidogrel response in cerebrovascular disease

Purpose

Clopidogrel is a prodrug that requires transformation into an active metabolite by cytochrome P450 (CYP) in the liver in order to irreversibly inhibit the P2Y12 adenosine diphosphate platelet receptor. CYP2C19 polymorphism has been reported to correlate with reduced antiplatelet activity of clopidogrel in coronary artery disease. We assessed the association between CYP2C19 polymorphism and clopidogrel resistance in patients with cerebrovascular disease.

Materials and Methods

We retrospectively gathered data from patients who experienced cerebrovascular disease, received clopidogrel, and were tested for clopidogrel resistance and CYP2C19 polymorphism. Clopidogrel resistance was tested by the VerifyNow P2Y12 system, and the CYP2C19 polymorphism was tested by the Seeplex CYP2C19 ACE Genotyping system. Clopidogrel resistance was expressed in P2Y12 reaction units (PRU) and percent inhibition. High PRU and low percent inhibition suggests clopidogrel resistance. CYP2C19 polymorphisms were expressed as extensive, intermediate, and poor metabolizers. Clopidogrel resistance was assessed according to the subgroup of CYP2C19 polymorphism.

Results

A total of 166 patients were evaluated. The PRU values of extensive CYP2C19 metabolizers (195.0±84.9) were significantly lower than those of intermediate and poor metabolizers (237.9±88.0, 302.2±58.9). The percent inhibition of extensive metabolizers (44.6±21.8) was significantly higher than that of intermediate and poor metabolizers (30.5±21.5, 14.0±13.4).

Conclusion

Intermediate and poor metabolizing CYP2C19 polymorphism is associated with reduced clopidogrel antiplatelet activity in patients with cerebrovascular disease. The clinical implications of this finding require further investigation.     

Resistance to antiplatelet drugs in patients with cerebrovascular disorders

This review concerns clinical and laboratory resistance to antiplatelet drugs (aspirin and clopidogrel) in patients with cerebrovascular disorders. Results of certain clinical trials showed that laboratory resistance to antiaggregants is associated with recurrent thromboembolic vascular events. The commonest causes of aspirin resistance are production of arachidonic acid metabolites via the lipoxygenase pathway, poor compliance with the treatment, polymorphism of the genes encoding for cyclooxygenase and glycoprotein (GP) IIb/IIIa, endothelial dysfunction. The causes of clopidogrel resistance include inadequate doses of the drug, its low absorption, poor compliance with the treatment, polymorphism of ADP receptors, GP IIb/IIIa and cytochrome P450 genes, acute coronary syndrome and stroke, metabolic syndrome. Therapeutic efficacy of antiaggregants can be improved by increasing their doses, using membranotropic agents, correcting endothelial dysfunction, etc. Because the apparent variability of antiplatelet drug resistance is currently due to the use of different test-systems by different authors, the evaluation of individual sensitivity to a given drug showing laboratory resistance and the choice of alternative therapy are thus far possible only in the framework of clinical studies. Large-scale prospective multicenter trials of antiplatelet drug resistance are needed along with research for better understanding mechanisms of individual platelet sensitivity and resistance to antiaggregants and developing efficacious methods for their correction.     

Arm Blood Flow and Oxygenation on the Transition from Arm to Combined Arm and Leg Exercise in Humans

The cardiovascular response to exercise with several groups of skeletal muscle implies that work with the legs may reduce arm blood flow. This study followed arm blood flow ( _arm) and oxygenation on the transition from arm cranking (A) to combined arm and leg exercise (A+L). Seven healthy male subjects performed A at ∼80 % of maximum work rate ( W _max) and A at ∼80 % W _max combined with L at ∼60 % W _max. A transition trial to volitional exhaustion was performed where L was added after 2 min of A. The _arm was determined by constant infusion thermodilution in the axillary vein and changes in biceps muscle oxygenation were measured with near-infrared spectroscopy. During A+L _arm was lowered by 0.38 ± 0.06 l min⁻¹ (10.4 ± 3.3 %,   P < 0.05  ) from 2.96 ± 1.54 l min⁻¹ during A. Total (HbT) and oxygenated haemoglobin (HbO₂) concentrations were also lower. During the transition from A to A+L _arm decreased by 0.22 ± 0.03 l min⁻¹ (7.9 ± 1.8 %,   P < 0.05  ) within 9.6 ± 0.2 s, while HbT and HbO₂ decreased similarly within 30 ± 2 s. At the same time mean arterial pressure and arm vascular conductance also decreased. The data demonstrate reduction in blood flow to active skeletal muscle during maximal whole body exercise to a degree that arm oxygen uptake and muscle tissue oxygenation are compromised.     

Purinergic regulation of epithelial transport

Purinergic receptors are a family of ubiquitous transmembrane receptors comprising two classes, P1 and P2 receptors, which are activated by adenosine and extracellular nucleotides (i.e. ATP, ADP, UTP and UDP), respectively. These receptors play a significant role in regulating ion transport in epithelial tissues through a variety of intracellular signalling pathways. Activation of these receptors is partially dependent on ATP (or UTP) release from cells and its subsequent metabolism, and this release can be triggered by a number of stimuli, often in the setting of cellular damage. The function of P2Y receptor stimulation is primarily via signalling through the G_q/PLC-β pathway and subsequent activation of Ca²⁺-dependent ion channels. P1 signalling is complex, with each of the four P1 receptors A₁, A_2A, A_2B, and A₃ having a unique role in different epithelial tissue types. In colonic epithelium the A_2B receptor plays a prominent role in regulating Cl⁻ and water secretion. In airway epithelium, A_2B and A₁ receptors are implicated in the control of Cl⁻ and other currents. In the renal tubular epithelium, A₁, A_2A, and A₃ receptors have all been identified as playing a role in controlling the ionic composition of the lumenal fluid. Here we discuss the intracellular signalling pathways for each of these receptors in various epithelial tissues and their roles in pathophysiological conditions such as cystic fibrosis.     

High CO2 chemosensitivity versus wide sensing spectrum: a paradoxical problem and its solutions in cultured brainstem neurons

CO₂ central chemoreceptors play an important role in cardiorespiratory control. They are highly sensitive to P _CO2 in a broad range. These two sensing properties seem paradoxical as none of the known pH-sensing molecules can achieve both. Here we show that cultured neuronal networks are likely to solve the sensitivity versus spectrum problem with parallel and serial processes. Studies were performed on dissociated brainstem neurons cultured on microelectrode arrays. Recordings started after a 3 week initial period of culture. A group of neurons were dose-dependently stimulated by elevated CO₂ with a linear response ranging from 20 to 70 Torr. The firing rate of some neurons increased by up to 30% in response to a 1 Torr P _CO2 change, indicating that cultured brainstem neuronal networks retain high CO₂ sensitivity in a broad range. Inhibition of Kir channels selectively suppressed neuronal responses to hypocapnia and mild hypercapnia. Blockade of TASK channels affected neuronal response to more severe hypercapnia. These were consistent with the p K _a values measured for these K⁺ channels in a heterologous expression system. The CO₂ chemosensitivity was reduced but not eliminated by blockade of presynaptic input from serotonin, substance P or glutamate neurons, indicating that both pre and postsynaptic neurons contribute to the CO₂ chemosensitivity. These results therefore strongly suggest that the physiological P _CO2 range appears to be covered by multiple sensing molecules, and that the high sensitivity may be achieved by cellular mechanisms via synaptic amplification in cultured brainstem neurons.     

IgE responses to Dermatophagoides pteronyssinus native major allergens Der p 1 and Der p 2 during long-term specific immunotherapy

We investigated by ELISA the IgE response to whole extract of the house-dust mite Dermatophagoides pteronyssinus (Dp) and to the native major allergens, Der p 1 and Der p 2, in sera from 18 adult patients (group A) with Dp-allergic asthma before ( t ₀) and 1, 2, 3, and 4 ( t ₁– t ₄) years after subcutaneous specific immunotherapy (SIT). A qualitative reduction ( P =0.05) of the IgE responses to Dp and Der p 2 was observed from t ₁ to t ₄, but a highly statistical significant decrease appeared at t ₃, ( P < 0.01). With regard to Der p 1 IgE values, the immunotherapy induced a significant decrease ( P < 0.01) at t ₃, but not before. In group A, the IgE responses to Der p 1 and Der p 2 were not correlated at t ₀ ( r _s=0.31; P = 0.2l) but were correlated at t₃ ( r _s= 0.78; P=0.001). We also examined sera from 14 adult patients (group B, same SIT schedule as group A) who were without respiratory symptoms at the end of the third year (t₃) of Dp SIT. At this time ( t ₃), there were no significant differences in Der p 1 and Der p 2 IgE levels between group A and group B.     

The bed nucleus of the stria terminalis modulates exercise-evoked cardiovascular responses in rats

Dynamic exercise evokes sustained cardiovascular changes, which are characterized by blood pressure and heart rate (HR) increases. Although it is well accepted that there is a central nervous system (CNS) mediation of cardiovascular adjustments during dynamic exercise, information on the role of specific CNS structures is limited. The bed nucleus of the stria terminalis (BST) is a forebrain structure known to be involved in central cardiovascular control. Based on this, we tested the hypothesis that BST modulates HR and mean arterial pressure (MAP) responses evoked when rats are submitted to dynamic exercise. Male Wistar rats were tested at three levels of exercise (0.4, 0.8 and 1 km h⁻¹) on a rodent treadmill before and after BST treatment with CoCl₂, a non-selective neurotransmission blocker. Bilateral microinjection of CoCl₂ (1 nmol in 100 nl artificial cerebrospinal fluid) into the BST reduced the pressor response to exercise at 0.4 km h⁻¹ as well as the tachycardic responses evoked by exercise at 0.4, 0.8 and 1 km h⁻¹. The BST treatment with CoCl₂ did not affect baseline MAP or HR, suggesting a lack of tonic BST influence on cardiovascular parameters at rest. Moreover, BST treatment with CoCl₂ did not affect motor performance in the open-field test, which indicates that effects of BST inhibition on cardiovascular responses to dynamic exercise are not due to changes in motor activity. The present results suggest that local neurotransmission in the BST modulates exercise-related cardiovascular adjustments. Data indicate that BST facilitates pressor and tachycardic responses evoked by dynamic exercise in rats.     

Tidal volume, cardiac output and functional residual capacity determine end-tidal CO2 transient during standing up in humans

In man assuming the upright position, end-tidal P _CO2 ( P _ETCO2 ) decreases. With the rising interest in cerebral autoregulation during posture change, which is known to be affected by P _ETCO2 , we sought to determine the factors leading to hypocapnia during standing up from the supine position. To study the contribution of an increase in tidal volume ( V _T ) and breathing frequency, a decrease in stroke volume (SV), a ventilation–perfusion ( V/Q ) gradient and an increase in functional residual capacity (FRC) to hypocapnia in the standing position, we developed a mathematical model of the lung to follow breath-to-breath variations in P _ETCO2 . A gravity-induced apical-to-basal V/Q gradient in the lung was modelled using nine lung segments. We tested the model using an eight-subject data set with measurements of V _T , pulmonary O₂ uptake and breath-to-breath lumped SV. On average, the P _ETCO2 decreased from 40 mmHg to 36 mmHg after 150 s standing. Results show that the model is able to track breath-to-breath P _ETCO2 variations ( r ²= 0.74, P < 0.05). Model parameter sensitivity analysis demonstrates that the decrease in P _ETCO2 during standing is due primarily to increased V _T , and transiently to decreased SV and increased FRC; a slight gravity-induced V/Q mismatch also contributes to the hypocapnia. The influence of cardiac output on hypocapnia in the standing position was verified in experiments on human subjects, where first breathing alone, and then breathing, FRC and V/Q were controlled.     

Effects of aspirin and clopidogrel versus oral anticoagulation on platelet function and on coagulation in patients with nonvalvular atrial fibrillation (CLAFIB)

The aim of the study was to evaluate the effect of two antithrombotic therapies on platelet function and on coagulation in patients with nonvalvular atrial fibrillation (NVAF). Twenty patients with NVAF were treated with aspirin (300 mg/day) and clopidogrel (75 mg/day) for 2 weeks immediately followed by oral anticoagulation (target international normalized ratio 2.0-3.0). Parameters of platelet function and coagulation were evaluated before antithrombotic therapy, at the end of aspirin plus clopidogrel and during subsequent anticoagulation treatment. Aspirin plus clopidogrel significantly inhibited platelet aggregation, fibrinogen receptor activation and release of P-selectin and prolonged in vitro bleeding time (p < 0.01). Coagulation parameters (platelet-dependent thrombin generation, antithrombin III, thrombin-antithrombin III complex, prothrombin fragment 1 + 2) were not significantly affected. During the subsequent oral anticoagulation phase platelet function was not substantially reduced; however, coagulation parameters were significantly inhibited (p < 0.001). The results indicate that combined antiplatelet therapy is superior to aspirin monotherapy in inhibiting platelet function but does not seem to substantially modulate coagulation cascade in patients with NVAF.     

硫化氢对FeCl_3诱导的小鼠颈动脉血栓模型中凝血和纤溶活性的影响

目的:探讨外源性硫化氢(H_2S)对三氯化铁(FeCl_3)诱导的小鼠颈动脉血栓模型凝血和纤溶活性的影响。方法:本实验分为空白对照组、模型组、不同浓度(12.5、25和50μmol/kg)的硫氢化钠(NaHS,H_2S供体)处理组和30 mg/kg氯吡格雷(clopidogrel,阳性对照)。不同浓度NaHS腹腔注射及硫酸氢氯吡格雷灌胃给药处理3d后,采用10%FeCl_3诱导小鼠颈动脉血栓模型。取各组颈动脉制成冰冻切片后,HE染色观察血栓形成及血管病理变化;对形成的血栓重量进行测量,计算血栓形成抑制率;采用血凝仪检测血浆凝血酶原时间(PT)、活化部分凝血活酶时间(APTF)、纤维蛋白原(FIB)和纤维蛋白降解产物(FDP)的变化;ELISA法检测血浆中血栓烷素B_2(TXB_2)、6-酮-前列腺素F_(1α)(6-keto-PGF_(1α))和纤溶酶原活化抑制剂(PAI)的含量。结果:与模型组相比,NaHS剂量依赖性地抑制小鼠颈动脉血栓的形成;NaHS处理可延长血栓模型中PT和APTT,减少FIB含量,增加FDP含量;NaHS处理能够降低颈动脉血栓模型中TXB_2及PAI含量,恢复6-keto-PGF_(1α)含量,并且6-keto-PGF_(1α)/TXB_2与血栓重量呈负相关。结论:硫化氢通过抑制机体凝血功能并激活纤溶活性而抑制FeCl_3诱导的颈动脉血栓形成。     

Facilitating roles of murine platelet glycoprotein Ib and αIIbβ3 in phosphatidylserine exposure during vWF–collagen-induced thrombus formation

Vessel wall damage exposes collagen fibres, to which platelets adhere directly via the collagen receptors glycoprotein (GP) VI and integrin α₂β₁ and indirectly by collagen-bound von Willebrand factor (vWF) via the GPIb-V-IX and integrin αIIbβ3 receptor complexes. Platelet–collagen interaction under shear stimulates thrombus formation in two ways, by integrin-dependent formation of platelet aggregates and by surface exposure of procoagulant phosphatidylserine (PS). GPVI is involved in both processes, complemented by α2β1. In mouse blood flowing over collagen, we investigated the additional role of platelet–vWF binding via GPIb and αIIbβ3. Inhibition of GPIb as well as blocking of vWF binding to collagen reduced stable platelet adhesion at high shear rate. This was accompanied by delayed platelet Ca²⁺ responses and reduced PS exposure, while microaggregates were still formed. Inhibition of integrin αIIbβ3 with JON/A antibody, which blocks αIIbβ3 binding to both vWF and fibrinogen, reduced PS exposure and aggregate formation. The JON/A effects were not enhanced by combined blocking of GPIb–vWF binding, suggesting a function for αIIbβ3 downstream of GPIb. Typically, with blood from FcR γ-chain +/− mutant mice, expressing 50% of normal platelet GPVI levels, GPIb blockage almost completely abolished platelet adhesion and PS exposure. Together, these data indicate that, under physiological conditions of flow, both adhesive receptors GPIb and αIIbβ3 facilitate GPVI-mediated PS exposure by stabilizing platelet binding to collagen. Hence, these glycoproteins have an assistant procoagulant role in collagen-dependent thrombus formation, which is most prominent at reduced GPVI activity and is independent of the presence of thrombin.     

Urinary leukotriene E4 and 11-dehydrothromboxane B2 in patients with aspirin-sensitive asthma

The objective of this study was to define the participation of cysteinyl leukotrienes (LTs) or thromboxane A₂ in the pathogenesis of aspirin-sensitive asthma (ASA). Leukotriene E₄ (LTE₄) and 11-dehydrothromboxane B₂ (11DTXB₂) values in spot urine were measured in 22 asthmatics with a history of aspirin sensitivity and in 17 without such a history of aspirin-sensitive asthma [NASA]) in the outpatient clinic. The urinary LTE₄ value was significantly higher in ASA patients than in NASA (340±47 vs 65±15 pg/mg·cr, P <0.001), but there was no significant difference in urinary 11DTXB2 between the two groups (891±77 vs 657±90 pg/mg·cr). A high value of LTE4 was not associated with type of asthma, severity of disease, oral prednisolone treatment, sex, or age. A higher value of 11DTXB₂ was observed in the atopic type than the nonatopic type in ASA (1086±111 vs 697±147 pg/mg·cr, P<0.05). No correlation was observed between urinary LTE₄ and 11DTXB₂ in either ASA or NASA. In conclusion, LTs may play an important role in the pathogenesis of ASA, and TXA₂ in the pathogenesis of the atopic type in ASA.     

Comparative vascular effects of histamine, prostaglandin (PG) D2 and its metabolite 9α, 11β-PGF2 in human skin

In this double-blind study we have investigated the vascular effects of prostaglandin, (PG) D₂, in normal skin and compared these effects with histamine and the initial PGD₂ metabolite 9α, 11β-PGF₂. In eight healthy subjects the vascular response to intradermal injections of histamine, PGD₂, a combination of histamine and PGD₂, and 9α, 11β-PGF₂, was assessed by measurement of the weal and flare area. Histamine caused dose-related increases in weal area ( P <0.01). The weal response due to PGD₂ was greater than saline control only at a dose of 71.0 and 710 nmol ( P <0.05). Because of the small size of the weal produced by PGD₂ when compared with histamine, it was not possible to determine their relative potencies. Histamine and PGD₂ caused dose-related increases in flare area ( P <0.05), and when compared at a response level of 10 cm² and 15 cm², histamine was 45 and 251 ( P <0.01) times more potent than PGD₂ in molar terms. Weal and flare responses due to 9α, 11β-PGF₂ were similar to those observed with the equimolar concentration of PGD₂. The weal and flare responses when PGD₂ and histamine when combined were not significantly different from that predicted by a purely additive effect. We conclude that histamine is likely to be an important mediator contributing towards increased vascular permeability and vasodilatation following immunological activation of skin mast cells in vivo , while PGD₂ and its metabolite 9α, 11β-PGF₂ play only a minor role.     

SUR2A C-terminal fragments reduce KATP currents and ischaemic tolerance of rat cardiac myocytes

C-terminal fragments of the sulphonylurea receptor SUR2A can alter the functional expression of cloned ATP-sensitive K⁺ channels (K_ATP). To investigate the protective role of K_ATP channels during metabolic stress we transfected SUR2A fragments into adult rat cardiac myocytes. A fragment comprising residues 1294–1358, the A-fragment, reduced sarcolemmal K_ATP currents by over 85% after 2 days (pinacidil-activated current densities were: vector alone 7.04 ± 1.22; and A-fragment 0.94 ± 0.07 pA pF⁻¹, n = 6,6, P < 0.001). An inactive fragment (1358–1545, current density 6.30 ± 0.85 pA pF⁻¹, n = 6) was used as a control. During metabolic inhibition (CN and iodoacetate) of isolated myocytes stimulated at 1 Hz, the A-fragment delayed action potential shortening and contractile failure, but accelerated rigor contraction and increased Ca²⁺ loading. On reperfusion, A-fragment-transfected cells also showed increased intracellular Ca²⁺ and the proportion of cells recovering contractile function was reduced from 40.0 to 9.5% ( P < 0.01). The protective effect of pretreatment with 2,4-dinitrophenol, measured from increased functional recovery and reduced Ca²⁺ loading, was abolished by the A-fragment. Our data are consistent with a role for K_ATP channels in causing action potential failure and reduced Ca²⁺ loading during metabolic stress, and with a major role in protection by preconditioning. The effects of the A-fragment may arise entirely from reduced expression of the sarcolemmal K_ATP channel, but we also discuss the possibility of mitochondrial effects.     

Fluid flow induces mechanosensitive ATP release, calcium signalling and Cl− transport in biliary epithelial cells through a PKCζ-dependent pathway

ATP in bile is a potent secretogogue, stimulating cholangiocyte Cl⁻ and fluid secretion via binding to membrane P2 receptors, though the physiological stimuli involved in biliary ATP release are unknown. The goal of the present studies was to determine the potential role of fluid flow in biliary ATP release and secretion. In both human Mz-Cha-1 biliary cells and normal rat cholangiocyte monolayers, exposure to flow increased relative ATP release which was proportional to the shear stress. In parallel studies, shear was associated with an increase in [Ca²⁺]_i and membrane Cl⁻ permeability, which were both dependent on extracellular ATP and P2 receptor stimulation. Flow-stimulated ATP release was dependent on [Ca²⁺]_i, exhibited desensitization with repetitive stimulation, and was regulated by PKCζ. In conclusion, both human and rat biliary cells exhibit flow-stimulated, PKCζ-dependent, ATP release, increases in [Ca²⁺]_i and Cl⁻ secretion. The finding that fluid flow can regulate membrane transport suggests that mechanosensitive ATP release may be a key regulator of biliary secretion and an important target to modulate bile flow in the treatment of cholestatic liver diseases.