Nerve growth factor in muscle afferent neurons of peripheral artery disease and autonomic function

In peripheral artery disease patients, the blood supply directed to the lower limbs is reduced. This results in severe limb ischemia and thereby enhances pain sensitivity in lower limbs. The painful perception is induced and exaggerate during walking, and is relieved by rest. This symptom is termed by intermittent claudication. The limb ischemia also amplifies autonomic responses during exercise. In the process of pain and autonomic responses originating exercising muscle, a number of receptors in afferent nerves sense ischemic changes and send signals to the central nervous system leading to autonomic responses. This review integrates recent study results in terms of perspectives including how nerve growth factor affects muscle sensory nerve receptors in peripheral artery disease and thereby alters responses of sympathetic nerve activity and blood pressure to active muscle. For the sensory nerve receptors, we emphasize the role played by transient receptor potential vanilloid type 1, purinergic P2X purinoceptor 3 and acid sensing ion channel subtype 3 in amplified sympathetic nerve activity responses in peripheral artery disease.     

Luminal ATP-induced contraction of rabbit pulmonary arteries and role of purinoceptors in the regulation of pulmonary arterial pressure

The effects of luminal ATP between rabbit pulmonary (PAs) and coronary arteries (CAs) were compared to understand the role of purinoceptors in the regulation of pulmonary arterial pressure (PAP) under hypoxia. Diameters of vessels were video analyzed under luminal perfusion. ATP-induced membrane currents and intracellular Ca²⁺ signals ([Ca²⁺]_i) were compared in pulmonary (PASMCs) and coronary myocytes (CASMCs) using patch clamp and spectrofluorimetry. PAP was measured in perfused lungs under ventilation. Luminal ATP induced constriction of rabbit PAs in the presence of endothelium. In contrast, CAs showed dilating responses to luminal ATP even in the absence of endothelium. In PASMCs, both P2X-mediated inward current and P2Y-mediated store Ca²⁺ release were consistently observed. In contrast, CASMCs showed neither P2X nor P2Y responses. In the perfused lungs, hypoxia-induced PAP increase was decreased by suramin, a purinergic antagonist. A luminal application of α,β-meATP largely increased PAP, whereas UTP decreased PAP. The combined application of P2X- and P2Y-selective agonists (α,β-meATP and UTP) increased PAP. However, the perfusion of ATP alone decreased PAP, and the ATP-induced PAP decrease was affected neither by adenosine receptor antagonist nor by nitric oxide synthase inhibitor. In summary, although the luminal ATP constricts isolated PAs and suramin attenuated the HPV of perfused lungs, the bimodal responses of PAP to purinergic agonists indicate that the luminal ATP regulates pulmonary circulation via complex signaling interactions in situ.     

ATP and Pain

Abstract: Many decades have passed since the pain‐producing properties of ATP were demonstrated in both animals and humans. ^{1 , 2} However, the more recent discovery of a family of ion channels for which ATP is a ligand and which are expressed by nociceptive neurons, has led to a resurgence of interest into the physiological and pathophysiological actions of ATP. This article considers the extent to which available evidence supports the notion that ATP receptors might be important novel analgesic targets. The hypothesis that ATP is a pain mediator is considered in terms of: the distribution of ATP receptors (specifically the P2X ion channel family); whether ATP release occurs under appropriate conditions; the evidence that ATP is capable of initiating pain in humans and pain‐related behaviour in animals; and, lastly, the analgesic effects of pharmacological or molecular block of ATP receptors.     

Fluid Flow Modulates Vascular Endothelial Cytosolic Calcium Responses to Adenine Nucleotides

Objective : To determine whether fluid flow influences the action of soluble vasoactive agonists on vascular endothelium. Methods : Confluent monolayers of bovine aortic endothelial cells (BAEC) were cultured on glass coverslips, prelabeled with the Ca²⁺-sensitive dye fura-2, and placed in a parallel-plate flow chamber designed to generate defined laminar fluid flow. Cytosolic free Ca²⁺ concentration ([Ca²⁺];) in individual BAEC was monitored during perfusion with medium containing adenine nucleotide under defined flow conditions. Results : Continuous perfusion with ATP (0.3–3.0 μM) or ADP (0.1–1.0 μM) evoked repetitive oscillations in [Ca²⁺]; in individual BAEC. The frequency of the [Ca²⁺]; oscillations was dependent on both nucleotide concentration and levels of applied shear stress; at constant bulk concentration of nucleotide, the frequency increased with shear stress. Stopping flow in the continuous presence of agonists immediately extinguished the oscillatory response. Elimination of extracellular Ca²⁺ did not inhibit the [Ca²⁺]; oscillations. In the presence of nonhydrolyzable nucleotide analog, ATPγS or ADPβS, application of flow resulted in similar shear-dependent [Ca²⁺]; oscillations, suggesting that flow modulation of the [Ca²⁺]; response was not simply due to depletion of ATP or ADP in the vicinity of BAEC monolayers as a result of hydrolysis of nucleotides by ectonucleotidases. Conclusions : These findings suggest that local hemodynamic conditions may modulate the action of vasoactive agents on the vascular endothelium in vivo.     

Role of ATP and Related Purine Compounds on Urethral Relaxation in Male Rabbits

Background Non-adrenergic, non-cholinergic (NANC) relaxation evoked by electrical field stimulation (EFS) has been observed in the urethra, with nitric oxide (NO) considered the agent most probably mediating this effect. However, Burnstock's purinergic hypothesis suggests that ATP and related purine compounds are neurotransmitters in NANC relaxation, although the physiological and pharmacological effects of ATP and related purine compounds in the urethra have been little studied. Methods The effects of ATP and related purine compounds, N_G-nitro-L -arginine (NOARG; an inhibitor of nitric oxide synthesis from l -arginine), calcitonin gene-related peptide (CGRP), substance P and vasoactive intestinal polypeptide (VIP) on relaxation and smooth muscle tension induced by electrical field stimulation (EFS) were studied in isolated male rabbit circular urethral smooth muscle (functional study). In addition, the outflow of ATP elicited by EFS was measured using the luciferase technique (superfusion study). All experiments were performed in the presence of guanethidine (3 times 10_-3 mol/L) and atropine (10_-6 mol/L). Results In preparations contracted with U46619, a prostaglandin peroxidase inhibitor, ATP had almost no effect on EFS-induced relaxation; however, suramin, a non-selective P₂₎-purinoceptor antagonist, and NOARG each markedly attenuated this relaxation in a concentration-dependent manner. ATP and related purine compounds (adenosine, AMP and ADP) each reduced U46619-induced tonic contraction in a concentration-dependent manner. The potencies of the relaxant effects of ATP and these purine compounds were almost the same. In preparations contracted with U46619, CGRP and substance P had no effect on tonic contraction, but VIP reduced tonic contraction in a concentration-dependent manner. In the superfusion study, the outflow of ATP into the superfusate was markedly increased by EFS. When NOARG or prazosin was added to the superfusate, the increase in outflow of ATP was unchanged, but when suramin was added to the superfusate, no increase in outflow of ATP was observed. Conclusions These findings suggest that P₂₎-purinoceptors exist in the male rabbit urethra, and that ATP and related purine compounds may play a role in non-adrenergic, non-cholinergic neurotransmission. Consequently, the pathways mediating urethral relaxation by ATP, NO and VIP may be different.     

Inhibition of NMDA-gated ion channels by the P2 purinoceptor antagonists suramin and reactive blue 2 in mouse hippocampal neurones

1. The action of suramin and reactive blue 2 on N-methyl-D-aspartate (NMDA)-activated ion current was studied in mouse hippocampal neurones in culture by use of whole-cell patch-clamp recording.
2. Suramin and reactive blue 2 inhibited steady-state current activated by 25 μM NMDA with IC₅₀ values of 68 and 11 μM, respectively.
3. Reactive blue 2 produced a gradual decline of NMDA-activated current to a steady-state, but this slow onset was not an indication of use-dependence, as it could be eliminated by exposure to reactive blue 2 before NMDA application. In addition, NMDA-activated current recovered completely from inhibition by reactive blue 2 in the absence of agonist.
4. The slow onset of inhibition by reactive blue 2 was not apparently due to an action at an intracellular site, as inclusion of 250 μM reactive blue 2 in the recording pipette did not alter inhibition by 25 μM reactive blue 2 applied externally.
5. Reactive blue 2 and suramin inhibited NMDA-gated channels in a voltage-independent manner.
6. Reactive blue 2, 25 μM, decreased the maximal response to NMDA from 1441 to 598 pA without changing its EC₅₀. In contrast, 75 μM suramin increased the EC₅₀ for NMDA from 13 to 35 μM, and decreased the maximal response to NMDA from 1822 to 1498 pA. Schild analysis of suramin inhibition of NMDA-activated current yielded a nonlinear plot.
7. Both agents decreased the maximal response to glycine without altering its EC₅₀.
8. Suramin and reactive blue 2 appear to inhibit NMDA receptor-channels in a manner that is noncompetitive with respect to both NMDA and glycine. However, inhibition by suramin differed from that by reactive blue 2, in that suramin significantly increased the EC₅₀ of NMDA.

     

P2X受体在大鼠舌乳头中的分布

研究用Ｐ２Ｘ１、Ｐ２Ｘ２,Ｐ２Ｘ３,Ｐ２Ｘ４,Ｐ２Ｘ５和Ｐ２Ｘ６受体的多克隆抗体,ＰＡＰ免疫组织化学及ＤＡＢ终产物金银加强法检查了大鼠味蕾和邻近组织中Ｐ２Ｘ受体的分布以探究ＡＴＰ在味觉激信号传导中的可能作用。结果：在轮廓砂、菌状乳头和叶状砂的味苗内显示出许多Ｐ２Ｘ３免疫反应阳性纤维,多呈含太,有的以味孔处。在在无味苗的粘膜上皮中,也发现少量Ｐ２Ｘ２免疫反应阳性神经纤维。在味蕾下的粘膜固有层中,近上     

Vasoactivity of diadenosine polyphosphates in human small renal resistance arteries.

BACKGROUND: We examined for the first time the vascular effects of purinergic agents that contribute to the regulation of peripheral vascular resistance in human small renal resistance arteries (hRRAs). METHODS AND RESULTS: Diadenosine polyphosphates (ApnAs, n = 3-6) and ATP, mounted in a microvessel myograph, caused vasoconstriction in hRRAs (rank order of potency: Ap5A > Ap6A = Ap4A > Ap3A = ATP). ADP, AMP and adenosine had less contractile potency than ApnA, suggesting that the observed effects were not induced by ApnA degradation products. The ApnA agent, Ap5A, but not Ap4A, induced vasoconstrictions that were inhibited by pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS; a P2X purinoceptor antagonist), but not by ADP3'5' (a P2Y purinoceptor antagonist). In pre-contracted hRRAs, all of the ApnA agents caused vasorelaxation, and the potencies did not differ from each other. The ApnA degradation products had less vasorelaxing potencies than ApnA, suggesting that the vasorelaxation was caused by the ApnA agents themselves. Ap4A-induced vasorelaxation was inhibited by ADP3'5' and PPADS. In contrast, Ap5A-induced vasorelaxation was not antagonized by ADP3'5', but was antagonized more strongly by PPADS than was Ap4A. CONCLUSIONS: We found that the tone of resistance arteries in human kidneys can be considerably influenced by these purinergic agonists, and most potently by ApnAs. Ap5A-induced vasoconstriction appeared to be mediated by P2X purinoceptors, whereas constriction due to Ap4A was caused by a different purinoceptor. Vasorelaxation due to Ap4A, but not Ap5A, appeared to be mediated by P2Y purinoceptors.     

ROLE OF BIVALENT CATIONS AND CHOLINE IN ATP-INDUCED APOPTOSIS OF HUMAN LYMPHOCYTESWITH P2Z RECEPTORS

The role of bivalent cations and choline in ATP induced apoptosis via P2Z purinceeptor was investigated in human leukemic lymphocytes. In vitro exposure of leukemic lymphocytes with P2Z receptors to 1mmol/L ATP or 0.1 mmol/L benzoylbenzoic ATP(13zATP) for 8 h in the presence of choline, 1 mmol/L Mg^2 or other bivalent cations, and ATP induced DNA breaks, associated with apoptosis were quantifie dby TdT assay. We observed that (l) Extracelhalar Mg^2 or Ca^2- stimulated ATP-indueed DNA fragmenration in a dose-depandent manner, and the compatihle evidence was provided by the inhibition of ATP-induced DNA fragmentation in the present of EGTA or EDTA; (2) ATP induced DNA fragmentation was completely inhibited by 1 mmol/L Zn^2 ; (3)ATP-induced DNA breaks were not afl[ected by Ba^2 , Sr^2 ,Co^2 when they were substituted for cxtracellular Mg^2 or Ca^2 ; (4)Chohne, an inhibitor of phospholipase D(PLD) stimulated by ATP through P2Z receptor in human lymphoeytes, was also a partial inhibitor of ATP-induced DNA fragmentation, and the results were coniqrmed by flow eytometrie analysis (FCA);(5)ATP induced DNA fragmentation was completely obliterated when the temperature was lower than 10℃. These results suggest that the endonuclease and PLD may be involved in ATP-induced apoptosis in human lymphocytes via P2Z receptor.     

A purinergic component of the excitatory postsynaptic current mediated by P2X receptors in the CA1 neurons of the rat hippocampus

The pyramidal neurons in the CA1 area of hippocampal slices from 17- to 19-day-old rats have been investigated by means of patch clamp. Excitatory postsynaptic currents (EPSCs) were elicited by stimulating the Schaffer collateral at a frequency below 0.2 Hz. It was found that inhibition of glutamatergic transmission by 20 μm 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 100 μm 2-amino-5-phosphonovaleric acid (D-APV) left a small component of the EPSC uninhibited. The amplitude of this residual EPSC (rEPSC) comprised 25 ± 11% of the total EPSC when measured at a holding potential of ?50 mV. The rEPSC was blocked by selective P2 blocker pyridoxal phosphate-6-azophenyl-2′-4′-disulphonic acid (PPADS) 10 μm and bath incubation with non-hydrolysable ATP analogues, ATP-γ-S and α,β-methylene-ATP at 50 and 20 μm , respectively. The rEPSC was dramatically potentiated by external Zn²⁺ (10 μm ). In another series of experiments exogenous ATP was applied to the CA1 neurons in situ. An inward current evoked by ATP was inhibited by PPADS to the same extent as the rEPSC. It is concluded that, depending on membrane voltage, about one-fifth to one-quarter of the EPSC generated by the excitatory synaptic input to the hippocampal CA1 neurons of rat is due to the activity of P2X receptors.