Actions of adenylyl compounds in the pedal disc of the cnidarian Actinia equina

1. The actions of adenylyl compounds were investigated in the circular muscle of the pedal disc of the sea anenome Actinia equina.2. Adenosine, adenosine 5'-diphosphate and adenosine 5'-triphosphate (ATP), but not adenosine 5'-monophosphate or the analogues of ATP, α,β-methylene ATP, and 2-methylthio ATP, caused concentration-dependent contractions.3. Neurogenic contractions in response to electrical field stimulation were not consistently affected by any of the adenylyl compounds and could be either potentiated or almost abolished by them.4. Reactive Blue 2, a vertebrate P₂-purinoceptor antagonist, caused concentration-dependent contractions which were mediated by nerves, being blocked by the anaesthetic chlorobutanol.5. The pharmacological profile of the adenylyl compounds in the pedal disc of Actinia equina is different from that observed in other invertebrate species and adds to the greater diversity of such profiles in invertebrates than in vertebrates.     

Intracellular signalling by nucleotide receptors in PC12 pheochromocytoma cells

The effect of extracellular ATP was studied in PC12 cells, a neurosecretory line that releases ATP. The addition of micromolar concentrations of ATP to PC12 cells evoked a transient increase in the cytosolic free Ca²⁺ concentration ([Ca²⁺]_i), as measured with the Ca²⁺-dye fura 2. AMP and adenosine were without effect, ruling out the involvement of P₁ receptors in mediating this response. The increase in [Ca²⁺]_i was reduced in calcium-free media and virtually eliminated by the addition of EGTA, suggesting that calcium influx was the primary response initiated by extracellular ATP. Nucleotide triphosphates such as UTP and, to a lesser degree, ITP also evoked an increase in [Ca²⁺]_i while GTP and CTP had little effect. In order to identify the receptor subtype mediating this response, the efficacy of ATP and ATP cogeners was assessed. The rank order potency was ATP > adenosine 5′-[γ-thio]triphosphate > ADP > 2-methylthioadenosine triphosphate (2-MeSATP) ～ adenosine 5′-[β-thio]diphosphate ? adenosine 5′-[αβ-methylene] triphosphate, adenosine 5′-[βγ-imido]triphosphate. This profile is not characteristic of either the P_2X or the conventional P_2Y receptors. The Ca²⁺ response exhibited desensitization to ATP that was dependent on the extracellular metabolism of ATP. UTP was equally effective in desensitizing the response. ATP, UTP, ITP, and to a much lesser extent 2MeSATP increased inositol phosphate production in a dose-dependent manner, suggesting receptor coupling to phosphatidylinositol-specific phospholipase C. These data are consistent with the view that PC12 cells express a class of non-P_2Y nucleotide receptors (P_2N) that mediate calcium influx and the accumulation of inositol phosphates. © 1993 Wiley-Liss, Inc.     

Influence of adenosine or adrenergic agonists on growth hormone stimulated lipolysis by chicken adipose tissue in vitro

In vitro lipolysis by chicken adipose explants was stimulated by growth hormone (GH) or glucagon. Adenosine or the adenosine agonist, N⁶-phenylisopropyladenosine (PIA), inhibited GH stimulated lipolysis, the effect of adenosine not being observed in the presence or adenosine deaminase. Glucagon induced lipolysis was also reduced by PIA. It is suggested that adenosine may act by G_i linked to either adenylate cyclase (for glucagon) or the signal transduction mechanism for GH. Lipolysis was not stimulated by GH in the presence of phenylephrine (α₁ adrenergic agonist), isoproterenol (β adrenergic agonist), adrenaline or glucagon. Although the presence of p-amino clonidine (α₂ adrenergic agonist) depressed basal lipolysis, a response to GH was still present. Either glucagon or β-adrenergic agonists (isoproterenol, adrenaline) stimulated lipolysis. In both cases, GH attenuated the lipolytic response to these hormones, which act via a cyclic adenosine monophosphate signal transduction mechanism.     

Identification of purinoceptors in the isolated stomach and intestine of the three-spined stickleback Gasterosteus aculeatus L.

1. Adenine nucleosides and nucleotides were examined for pharmacological activity in isolated stomach and intestine from the stickleback Gasterosteus aculeatus L.2. Adenosine and its stable analogues all concentration-dependently relaxed carbachol-contracted stomach and intestine, with no significant difference in the potency of the analogues. Only 8-(p-sulphophenyl) theophylline inhibited the relaxant response to adenosine in both tissues; other adenosine antagonists such as 1,3-dipropyl-8-cyclopentylxanthine were not active.3. ATP, α, β-methylene ATP (α,β-MeATP) and 2-methylthio ATP (2-MeSATP) all caused concen- tration-dependent contractions of the stomach and intestine.4. In the stomach, the order of potency was 2-MeSATP >α, β-MeATP = ATP; the P_2γ-purinoceptor antagonist reactive blue 2 inhibited responses to ATP.5. In the intestine, the order of potency was α,β-MeATP > 2-MeSATP = ATP; reactive blue 2 did not affect responses to ATP, nor did prolonged incubation with α,β-MeATP.6. It is concluded that in both the stomach and intestine, adenosine is acting through a non-specific or undifferentiated P₁-purinoceptor. In the stomach, however, the P₂-purinoceptor appears to be analogous to the mammalian P_2γ-purinoceptor, and in the intestine, the receptor is more similar to the mammalian P_2x-subtype, although it was not susceptible to desensitization.     

Purinergic modulation of spontaneous activity and of responses to high potassium and acetylcholine in rat ileal smooth muscle

1. In rat ileal smooth muscle both adenosine and ATP at 10⁻⁴ M significantly enhanced spontaneous mechanical activity. The excitatory actions of adenosine were blocked by the P₁ receptor antagonist 8-phenyltheophylline and the excitatory effects of ATP were significantly reduced by the P₂ receptor antagonist quinidine.2. The P₂ receptor desensitizer α,β-methylene-ATP was without effect on ACh responses nor did the stable analogue β,gg-methylene-ATP exert any effect on spontaneous mechanical activity.3. Pretreatment with adenosine caused a dose-dependent enhancement of K-induced contractures in the ileum. Low adenosine concentrations slightly inhibited and high concentrations slightly enhanced ACh-induced contractures in the ileum.4. ATP potentiated the phasic component of the ileal K-induced contracture but strongly inhibited tonic force at high concentrations. This agent slightly inhibited the phasic component of the ACh-induced contracture while strongly inhibiting ACh-induced tonic force.5. α,β-methylene-ATP inhibited ileal muscle ACh induced contractures while it potentiated both phasic and tonic K-induced contractures. β, γ-methylene ATP inhibited ACh-induced contractures but it enhanced K-induced phasic contractures while inhibiting K-induced tonic force.6. The results of this study suggest that rat ileum may contain the A₁ subtype of the P₁ receptor but the evidence for a P₂ receptor subtype is conflicting despite the inhibition of ATP actions by quinidine.7. The inhibition of K- and ACh-induced tonic force suggests that adenosine and ATP interactions with ileal smooth muscle may inactivate slow voltage-dependent calcium channels leading to EC uncoupling.     

Impairment of ATP hydrolysis decreases adenosine A1 receptor tonus favoring cholinergic nerve hyperactivity in the obstructed human urinary bladder

This study was designed to investigate whether reduced adenosine formation linked to deficits in extracellular ATP hydrolysis by NTPDases contributes to detrusor neuromodulatory changes associated with bladder outlet obstruction in men with benign prostatic hyperplasia (BPH). The kinetics of ATP catabolism and adenosine formation as well as the role of P1 receptor agonists on muscle tension and nerve-evoked [³H]ACh release were evaluated in mucosal-denuded detrusor strips from BPH patients (n = 31) and control organ donors (n = 23). The neurogenic release of ATP and [³H]ACh was higher (P < 0.05) in detrusor strips from BPH patients. The extracellular hydrolysis of ATP and, subsequent, adenosine formation was slower (t_1/2 73 vs. 36 min, P < 0.05) in BPH detrusor strips. The A₁ receptor-mediated inhibition of evoked [³H]ACh release by adenosine (100 μM), NECA (1 μM), and R-PIA (0.3 μM) was enhanced in BPH bladders. Relaxation of detrusor contractions induced by acetylcholine required 30-fold higher concentrations of adenosine. Despite VAChT-positive cholinergic nerves exhibiting higher A₁ immunoreactivity in BPH bladders, the endogenous adenosine tonus revealed by adenosine deaminase is missing. Restoration of A₁ inhibition was achieved by favoring (1) ATP hydrolysis with apyrase (2 U mL⁻¹) or (2) extracellular adenosine accumulation with dipyridamole or EHNA, as these drugs inhibit adenosine uptake and deamination, respectively. In conclusion, reduced ATP hydrolysis leads to deficient adenosine formation and A₁ receptor-mediated inhibition of cholinergic nerve activity in the obstructed human bladder. Thus, we propose that pharmacological manipulation of endogenous adenosine levels and/or A₁ receptor activation might be useful to control bladder overactivity in BPH patients.     

The NLRP3 inflammasome is activated by nanoparticles through ATP,ADP and adenosine

The NLR pyrin domain containing 3 (NLRP3) inflammasome is a major component of the innate immune system, but its mechanism of activation by a wide range of molecules remains largely unknown. Widely used nano-sized inorganic metal oxides such as silica dioxide (nano-SiO₂) and titanium dioxide (nano-TiO₂) activate the NLRP3 inflammasome in macrophages similarly to silica or asbestos micro-sized particles. By investigating towards the molecular mechanisms of inflammasome activation in response to nanoparticles, we show here that active adenosine triphosphate (ATP) release and subsequent ATP, adenosine diphosphate (ADP) and adenosine receptor signalling are required for inflammasome activation. Nano-SiO₂ or nano-TiO₂ caused a significant increase in P2Y1, P2Y2, A2_A and/or A2_B receptor expression, whereas the P2X7 receptor was downregulated. Interestingly, IL-1β secretion in response to nanoparticles is increased by enhanced ATP and ADP hydrolysis, whereas it is decreased by adenosine degradation or selective A2_A or A2_B receptor inhibition. Downstream of these receptors, our results show that nanoparticles activate the NLRP3 inflammasome via activation of PLC-InsP3 and/or inhibition of adenylate cyclase (ADCY)-cAMP pathways. Finally, a high dose of adenosine triggers inflammasome activation and IL-1β secretion through adenosine cellular uptake by nucleotide transporters and by its subsequent transformation in ATP by adenosine kinase. In summary, we show for the first time that extracellular adenosine activates the NLRP3 inflammasome by two ways: by interacting with adenosine receptors at nanomolar/micromolar concentrations and through cellular uptake by equilibrative nucleoside transporters at millimolar concentrations. These findings provide new molecular insights on the mechanisms of NLRP3 inflammasome activation and new therapeutic strategies to control inflammation.The inflammasome is a major factor of the innate immune system acting as a multiprotein platform to activate caspase-1. We showed recently that nanoparticles of TiO₂ (nano-TiO₂) and SiO₂ (nano-SiO₂) are sensed by the NLRP3 inflammasome to induce the release of mature IL-1β,¹ as observed previously with the environmental irritants asbestos or silica.² Despite the identification and characterisation of numerous sterile or microbial activators, the precise mechanisms mediating NLRP3 inflammasome activation remain to be determined. Here, we investigated whether ATP release and purinergic signalling through ATP, ADP and adenosine may be involved in inflammasome activation by nanoparticles. Intracellular ATP is released after cellular stress and/or activation, and purinergic signalling has been shown to modulate inflammation and immunity.^{3, 4} In the extracellular space, ATP is rapidly hydrolysed in a stepwise manner to ADP, AMP (adenosine monophosphate) and adenosine by ectoenzymes.⁴ Adenosine is then irreversibly hydrolysed to inosine by adenosine deaminase (ADA). Extracellular ATP (eATP) signals through both ATP-gated ion channels P2X and G protein-coupled receptor (GPCR) P2Y membrane receptors, whereas ADP signals through P2Y receptors and adenosine through P1 receptors (or A receptors).⁵ P2Y receptors and A receptors may be coupled to the G_q protein, which activates phospholipase C-beta (PLC-β), to the stimulatory G (G_s) protein, which stimulates adenylate cyclase inducing an increase in cyclic AMP (cAMP) levels, or to the G inhibitory (G_i) protein, which inhibits adenylate cyclase. Extracellular adenosine level is the result of adenosine production from extracellular ATP and ADP, its degradation into inosine and its reuptake by cells. Both ATP and adenosine can be transported outside of the cell via diffusion or active transport, whereas only adenosine can enter the cells through adenosine transporters.⁶ Most cells possess equilibrative and concentrative adenosine transporters (respectively, ENTs and CNTs), which allow adenosine to quickly cross the plasma membrane.⁷ Intracellular adenosine is converted to ATP via phosphorylation steps mediated by adenosine kinase (AK) and AMP kinase (AMPK). The basal physiological level of extracellular adenosine has been estimated to be in the range of 30–200 nM.⁸ ATP-derived adenosine and its subsequent signalling through P1 receptors have beneficial roles in acute disease states.^{4, 9} However, during tissue injury, elevated adenosine levels participate in the progression to chronic diseases by promoting aberrant wound healing leading to fibrosis in different organs including the lungs, liver, skin and kidney. In these conditions the blockade of adenosine signalling is beneficial.^{10, 11, 12, 13, 14, 15, 16} In murine models, ADA-knockout mice present high persistent adenosine levels, which lead to airspace enlargement and fibrosis, cardinal signs of COPD and IPF.^{14, 17, 18}Here we investigate in more detail the critical contribution of purinergic signalling in driving NLRP3 inflammasome activation in response to nanoparticles pointing out the effect of ATP, ADP, as well as adenosine and its receptors. We also identify ATP-derived adenosine as a potential activator of the inflammasome.     

Effects of purine compounds and forskolin on melanophores of the medaka,oryzias latipes: Evidence for an A2-adenosine receptor

1. The effects of purines on denervated melanophores of the medaka were studied under experimental conditions in which melanosomes were aggregated by norepinephrine or lithium ion beforehand.2. Adenosine and its derivatives caused melanosome dispersion; the order of potency for the series was; NECA > adenosine > ATP > 2-chloroadenosine > PIA > CHA > cyclic AMP.3. 8-Phenyltheophylline, a potent purinoceptor antagonist, blocked the effect of purines and caused a rightward shift of the adenosine and analog concentration-response curves.4. 8-Br cyclic AMP also caused melanosome dispersion but its action was not blocked by 8-phenyladenosine. Dibutyryl cyclic AMP, cyclic GMP, dibutyryl cyclic GMP, and 8-br cyclic GMP were all ineffective.5. The effect of adenosine was immediately eliminated by adenosine deaminase but, actions of NECA, AMP, ADP, ATP, and cyclic AMP were not.6. Forskolin, a potent activator of adenylate cyclase, mimicked the action of adenosine.7. It is concluded that adenosine and its derivatives mediate their melanosome-dispersing effect via a P₁-purinoceptor that displays characteristics of the A₂-subtype and that adenine nucleotides directly activate the A₂-receptor without conversion to adenosine.     

Hyperpolization-activated Ca channels in guard cell plasma membrane are involved in extracellular ATP-promoted stomatal opening in Vicia faba

Extracellular ATP (eATP) plays essential roles in plant growth, development, and stress tolerance. Extracellular ATP-regulated stomatal movement of Arabidopsis thaliana has been reported. Here, ATP was found to promote stomatal opening of Vicia faba in a dose-dependent manner. Three weakly hydrolysable ATP analogs (adenosine 5′-O-(3-thio) triphosphate (ATPγS), 3′-O-(4-benzoyl) benzoyl adenosine 5′-triphosphate (Bz-ATP) and 2-methylthio-adenosine 5′-triphosphate (2meATP)) showed similar effects, indicating that ATP acts as a signal molecule rather than an energy charger. ADP promoted stomatal opening, while AMP and adenosine did not affect stomatal movement. An ATP-promoted stomatal opening was blocked by the NADPH oxidase inhibitor diphenylene iodonium (DPI), the reductant dithiothreitol (DTT) or the Ca²⁺ channel blockers GdCl₃ and LaCl₃. A hyperpolarization-activated Ca²⁺ channel was detected in plasma membrane of guard cell protoplast. Extracellular ATP and weakly hydrolyzable ATP analogs activated this Ca²⁺ channel significantly. Extracellular ATP-promoted Ca²⁺ channel activation was markedly inhibited by DPI or DTT. These results indicated that eATP may promote stomatal opening via reactive oxygen species that regulate guard cell plasma membrane Ca²⁺ channels.     

Indirect effects of adenosine triphosphate on chloride secretion in mammalian colon

Summary The effects of adenosine triphosphate (ATP) on shortcircuit current (SCC) in rat colonic epithelium are described. ATP caused a large increase in inward-going current and was considerably more potent in this respect than ADP. AMP or adenosine. The response to ATP was sided, there being only minor effects when the nucleotide was added to the apical side of the tissue. The effects of ATP were not modified by the cyclooxygenase inhibitor, indomethacin, eliminating eicosanoid formation as a mechanism. The effects of ATP were potentiated by theophylline and not blocked by ,-methylene ATP. The data are consistent with the effect being dependent on the activation of adenylate cyclase, but it has not been possible to classify the receptors intoP ₁ orP ₂ categories. Using inhibitors of NaCl cotransport (piretanide), carbonic anhydrase (acetazolamide), and chloride channels (diphenylamine-2-carboxylate), it was concluded that the SCC response to ATP was due to chloride secretion with, perhaps, a minor contribution from bicarbonate. Flux measurements with²²Na and³⁶Cl confirmed this view, there being approximate equivalence of chloride secretion with the SCC responses. Additionally, flux measurements revealed an inhibition of electroneutral NaCl absorption in response to ATP.The effects of ATP were antagonized by tetrodotoxin (TTX), greater than 50% inhibition being achieved with 10nm TTX. This result suggests that ATP does not act directly on receptors in the epithelial cells but rather on neuronal elements in the lamina propria. It will be necessary to re-examine other secretagogues for indirect effects of this kind and to search for the final effector neurotransmitter which evokes secretion.     

Purinergic agonists stimulate the secretion of endothelin-1 in rat thyroid FRTL-5 cells

The aim of the present study was to investigate the mechanisms regulating endothelin-1 (ET-1) secretion in rat thyroid FRTL-5 cells. ET-1 was found to be secreted after stimulation with adenosine and ATP. The release of ET-1 was sensitive to pertussis toxin, indicating a role of G-proteins in the stimulus-secretion coupling. The stimulation evoked by ATP or adenosine was inhibited by the P₁-receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), and in the presence of adenosine deaminase the adenosine- and ATP-mediated ET-1 secretion was abolished. These evidences suggest a role of a P₁-adenosine receptor in the secretion of ET-1. Increasing cyclic AMP with forskolin decreased the adenosine-mediated secretion. In addition, the intracellular calcium chelator BAPTA or inhibition of calcium entry with Ni²⁺ prevented the response. Protein kinase C (PKC) is also partly involved in ET-1 secretion in FRTL-5 cells. Activation of PKC with the phorbol ester phorbol 12-myristate 13-acetate (PMA) stimulated the secretion of ET-1 in a time- and dose-dependent manner. Furthermore, downregulation of PKC decreased the secretion of ET-1 stimulated by adenosine. In conclusion, ET-1 secretion in FRTL-5 cells is stimulated via a pertussis toxin-sensitive P₁-receptor pathway which is modulated by several signal transduction mechanisms including cAMP, Ca²⁺, and PKC. © 1996 Wiley-Liss, Inc.     

Two Distinct P2 Purinergic Receptors, P2Y and P2U, Are Coupled to Phospholipase C in Mouse Pineal Gland Tumor Cells

Abstract: We found that extracellular ATP can increase the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in mouse pineal gland tumor (PGT-β) cells. Studies of the [Ca²⁺]_i rise using nucleotides and ATP analogues established the following potency order: ATP, adenosine 5′-O-(3-thiotriphosphate) ≥ UTP > 2-chloro-ATP > 3′-O-(4-benzoyl)benzoyl ATP, GTP ≥ 2-methylthio ATP, adenosine 5′-O-(2-thiodiphosphate) (ADPβS) > CTP. AMP, adenosine, α,β-methyleneadenosine 5′-triphosphate, β,γ-methyleneadenosine 5′-triphosphate, and UMP had little or no effect on the [Ca²⁺]_i rise. Raising the extracellular Mg²⁺ concentration to 10 mM decreases the ATP-and UTP-induced [Ca²⁺]_i rise, because the responses depend on the ATP^4? and UTP^4? concentrations, respectively. The P_2U purinoceptor-selective agonist UTP and the P_2Y purinoceptor-selective agonist ADPβS induce inositol 1,4,5-trisphosphate generation in a concentration-dependent manner with maximal effective concentrations of ～100 µM. In sequential stimulation, UTP and ADPβS do not interfere with each other in raising the [Ca²⁺]_i. Costimulation with UTP and ADPβS results in additive inositol 1,4,5-trisphosphate generation to a similar extent as is achieved with ATP alone. Pretreatment with pertussis toxin inhibits the action of UTP and ATP by maximally 45–55%, whereas it has no effect on the ADPβS response. Treatment with 1 µM phorbol 12-myristate 13-acetate inhibits the ADPβS-induced [Ca²⁺]_i rise more effectively than the ATP- and UTP-induced responses. These results suggest that P_2U and P_2Y purinoceptors coexist on PGT-β cells and that both receptors are linked to phospholipase C.     

Adenosine transport by cultured glial cells from chick embryo brain

The transport of adenosine was studied in pure cultures of glial cells from chick embryo brain. In order to avoid complications in uptake measurements due to adenosine metabolism, cultures were depleted of ATP by incubation with cyanide and iodoacetate prior to addition of [³H]adenosine. Under the 5- to 25-s periods used for the transport assay, no adenosine metabolism could be detected. Initial rates of adenosine transport under these conditions obeyed the Michaelis-Menten relationship with K_m = 370 μM and V_max = 10.3 nmol/min/mg cell protein. ATP depletion or elimination of Na⁺ from the assay medium had no significant effect on initial rates of adenosine uptake. However, when assays were carried out under conditions of significant adenosine metabolism (10-min uptake in the absence of metabolic inhibitors), a high-affinity incorporation process could be demonstrated in the glial cells (K_m = 12 μM; V_max = 0.34 nmol/ min/mg protein). The transport activity expressed in ATP-depleted glial cells was most sensitive to inhibition by nitrobenzylthioinosine, dipyridamole, and N⁶-benzyladenosine. In decreasing order of potency, N⁶-methyladenosine, 2-chloroadenosine, inosine, and thymidine also blocked adenosine translocation in glial cultures. Thus, adenosine transport by cultured glial cells occurs by means of a low-affinity, facilitated diffusion system which is similar to the nucleoside transporter in cells of nonneural origin.     

Low affinity purinergic receptor modulates the response of rat submandibular glands to carbachol and substance P

The effect of extracellular ATP on the intracellular calcium concentration ([Ca²⁺]_i) in rat submandibular glands was tested. The dose-response curve for ATP was biphasic with a first increase in the 1–30 μM concentration range and a further increase at concentrations higher than 100 μM. Among ATP analogs, only benzoyl-ATP stimulated the low affinity component. ATPτS blocked this response. All the other analogs tested reproduced the high-affinity low capacity response. Magnesium and Coomassie blue selectively blocked the low affinity component. High concentrations of ATP blocked the increase of the intracellular calcium concentration [Ca²⁺]_i in response to 100 μM carbachol. By itself, substance P (100 pM-1 μM) increased the [Ca²⁺]_i. One mM ATP potentiated the response to concentrations of substance P higher than 10 nM. This potentiation was reversed by extracellular magnesium. Carbachol 100 μM and substance P (100 pM-1 μM) increased the release of inositol trisphosphate (IP₃) from polyphosphoinositides (polyPI). Activation of the low affinity ATP receptors did not activate the polyPI-specific phospholipase C but inhibited its activation by 100 μM carbachol (−50%) and by 100 nM substance P (−60% at 1 nM substance P and −40% at 100 nM substance P). Substance P induced a strong homologous desensitization: a preincubation with 1 nM substance P nearly completely abolished the response to 1 μM substance P. When the cells were exposed to ATP before the second addition of substance P, the purinergic agonist partially restored the response to the tachykinin without totally reversing the desensitization. It is concluded that two types of purinergic receptors coexist in rat submandibular glands; a high-affinity, low capacity receptor which remains pharmacologically and functionally undefined and a low affinity site, high capacity receptor of the P_2Z type coupled to a non-selective cation channel. The occupancy of these low affinity sites blocks the increase of the [Ca²⁺]_i in response to a muscarinic agonist and the activation of polyPI-specific phospholipase C by carbachol and substance P. It potentiates the effect of high concentrations of substance P on the [Ca²⁺]_i. © 1996 Wiley-Liss, Inc.     

Energy dependent changes in the cytochromes of the mitochondrial respiratory chain

In intact mitochondria a stoichiometric coupling exists between cytochrome a₃ and the hydrolysis of adenosine triphosphate (ATP). In each case the modification of one cytochrome a₃ (measured as a spectral change) is coupled to the hydrolysis of one ATP molecule. When both cytochromes a₃ and a are reduced the measured equilibrium constant is 0.06 m^?1 but this constant is 10³ M^?1 when both cytochromes are oxidized. When the sixth ligand for cytochrome a₃ is an externally added ligand (HCN, H₂S, CO, NO) the equilibrium constant is different for each ligand, suggesting that the ATP induced modification is of the fifth ligand but that it is energetically dependent on the chemical nature of the sixth ligand. The measured half-reduction potentials for cytochromes a₃ and b_T are dependent on the concentrations of added ATP, adenosine diphosphate (ADP), and orthophosphate. The relationship is consistent with a ligand exchange mechanism in which the ligand on the cytochrome is dependent on the phosphate potential ($\text{ATP}\text{ADP}$ × P_i). The equilibrium constants obtained by the ligand exchange treatment of the E_m values for cytochrome a₃ are consistent with those obtained by direct measurement of the equilibrium constants for the spectrally measured changes.     

Proton efflux through the chloroplast ATP synthase (CF0 · CF1) in the presence of sulfhydryl-modifying agents

The rate of photosynthetic electron transport measured in the absence of ADP and P_i is stimulated by low levels of Hg²⁺ or Ag⁺ (50% stimulation ≈ 3 Hg²⁺ or 6 Ag⁺/100 chlorophyll) to a plateau equal to the transport rate under normal phosphorylating conditions (i.e. +ADP, +P_i). Chloroplasts pretreated in the light under energizing conditions with N-ethylmaleimide show a similar stimulation of non-phosphorylating electron transport. The stimulations of non-phosphorylating electron transport by Hg²⁺, Ag⁺ and N-ethylmaleimide are reversed by the CF₁ inhibitor phlorizin, the CF₀ inhibitor triphenyltin chloride, and can be further stimulated by uncouplers such as methylamine. The Hg²⁺ and N-ethylmaleimide stimulations, but not the Ag⁺ stimulation, are completely reversed by low levels of ADP (2 μM), ATP (2 μM), and P_i (400 μM). Ag⁺, which is a potent inhibitor of ATP synthesis, has little or no effect upon phosphorylating electron transport (+ADP, +P_i). Concomitant with the stimulations of non-phosphorylating electron transport by Hg²⁺, Ag⁺ and ADP + P_i, there is a decrease in the level of membrane energization (as measured by atebrin fluorescence quenching) which is reversed when the CF₀ channel is blocked by triphenyltin. These results suggest that modification of critical CF₁ sulfhydryl residues by Hg²⁺, Ag⁺ or N-ethylmaleimide leads to the loss of intra-enzyme coupling between the transmembrane protontransferring and the ATP synthesis activities of the CF₀-CF₁ ATP synthase complex.     

Formononetin,an isoflavone,relaxes rat isolated aorta through endothelium-dependent and endothelium-independent pathways

We evaluated the vasorelaxation effects of formononetin, an isoflavone/phytoestrogen found abundantly in Astragalus mongholicus Bunge, on rat isolated aorta and the underlying mechanisms involved. Cumulative administration of formononetin, genistein, daidzein and biochanin A relaxed phenylephrine-preconstricted aorta. Formononetin and biochanin A caused a similar magnitude of relaxation whereas daidzein was least potent. Mechanical removal of endothelium, L-NAME (100 μM) and methylene blue (10 μM) suppressed formononetin-induced relaxation. Formononetin increased endothelial nitric oxide (NO) synthase (eNOS), but not inducible NO synthase, activity with an up-regulation of eNOS mRNA and p-eNOS^Ser1177 protein expression. In endothelium-denuded preparations, formononetin-induced vasorelaxation was significantly reduced by glibenclamide (3 μM) and iberiotoxin (100 nM), and a combination of glibenclamide (3 μM) plus iberiotoxin (100 nM) abolished the relaxation. In contrast, formononetin-elicited endothelium-independent relaxation was not altered by ICI 182,780 (10 μM, an estrogen receptor (ERα/ERβ) antagonist) or mifepristone (10 μM, a progesterone receptor antagonist). In single aortic smooth muscle cells, formononetin caused opening of iberiotoxin-sensitive Ca²⁺-activated K⁺ (BK_Ca) channels and glibenclamide-sensitive adenosine triphosphate (ATP)-dependent K⁺ (K_ATP) channels. Thus, our results suggest that formononetin caused vascular relaxation via endothelium/NO-dependent mechanism and endothelium-independent mechanism which involves the activation of BK_Ca and K_ATP channels.     

Characterization of the membrane bound Mg2+-ATPase of rat skeletal muscle

A procedure was developed to isolate a membrane fraction of rat skeletal muscle which contains a highly active Mg²⁺-ATPase (5–25 μmol P_i/mg min). The rate of ATP hydrolysis by the Mg²⁺-ATPase was nonlinear but decayed exponentially (first-order rate constant ≥0.2 s^?1 at 37°C). The rapid decline in the ATPase activity depended on the presence of ATP or its nonhydrolyzable analog 5′-adenylyl imidodiphosphate (AdoPP[NH]P). Once inactivated, removal of ATP from the medium did not immediately restore the original activity. ATP- or AdoPP[NH]P-dependent inactivation could be blocked by concanavalin A, wheat germ agglutinin or rabbit antiserum against the membrane. Additions of these proteins after ATP addition prevented further inactivation but did not restore the original activity. Low concentrations of ionic and nonionic detergents increased the rate of ATP-dependent inactivation. Higher concentrations of detergents, which solubilize the membrane completely, inactivated the Mg²⁺-ATPase. Cross-linking the membrane components with glutaraldehyde prevented ATP-dependent inactivation and decreased the sensitivity of the Mg²⁺-ATPase to detergents. It is proposed that the regulation of the Mg²⁺-ATPase by ATP requires the mobility of proteins within the membrane. Cross-linking the membrane proteins with lectins, antiserum or glutaraldehyde prevents inactivation; increasing the mobility with detergents accelerates ATP-dependent inactivation.     

Pannexin 1 Channels Link Chemoattractant Receptor Signaling to Local Excitation and Global Inhibition Responses at the Front and Back of Polarized Neutrophils

Neutrophil chemotaxis requires excitatory signals at the front and inhibitory signals at the back of cells, which regulate cell migration in a chemotactic gradient field. We have previously shown that ATP release via pannexin 1 (PANX1) channels and autocrine stimulation of P2Y₂ receptors contribute to the excitatory signals at the front. Here we show that PANX1 also contributes to the inhibitory signals at the back, namely by providing the ligand for A_2A adenosine receptors. In resting neutrophils, we found that A_2A receptors are uniformly distributed across the cell surface. In polarized cells, A_2A receptors redistributed to the back where their stimulation triggered intracellular cAMP accumulation and protein kinase A (PKA) activation, which blocked chemoattractant receptor signaling. Inhibition of PANX1 blocked A_2A receptor stimulation and cAMP accumulation in response to formyl peptide receptor stimulation. Treatments that blocked endogenous A_2A receptor signaling impaired the polarization and migration of neutrophils in a chemotactic gradient field and resulted in enhanced ERK and p38 MAPK signaling in response to formyl peptide receptor stimulation. These findings suggest that chemoattractant receptors require PANX1 to trigger excitatory and inhibitory signals that synergize to fine-tune chemotactic responses at the front and back of neutrophils. PANX1 channels thus link local excitatory signals to the global inhibitory signals that orchestrate chemotaxis of neutrophils in gradient fields.     

Ca2+ responses to acetylcholine and adenosine triphosphate in the otocyst of chick embryo

The action of acetylcholine and adenosine triphosphate (ATP) on cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) was studied in the otocyst epithelium of embryonic day 3 chicks with Ca²⁺-sensitive fluorescence measurements. Increases in [Ca²⁺]_i were evoked by the bath application of acetylcholine (1 μM or higher). The rise in [Ca²⁺]_i was due to the release of Ca²⁺ from intracellular Ca²⁺ stores, since the Ca²⁺ response occurred even in a Ca²⁺-free medium. The Ca²⁺ response to acetylcholine was mediated by muscarinic receptors. Atropine of 1 μM abolisehd the response to 10 μM acetylcholine; muscarine and carbamylcholine (100 μM each) evoked Ca²⁺ rises. Increases in [Ca²⁺]_i were also evoked by the bath application of ATP (10 μM or higher). The Ca²⁺ rise by ATP was evoked even in a Ca²⁺-free medium. Adenosine (500 μM) did not cause any Ca²⁺ response. Suramin and reactive blue 2 (200 μM each) completely blocked the Ca²⁺ response to 500μM ATP. Uridine triphosphate (500 μM) caused comparable Ca²⁺ responses with those to 500 μM ATP. These results suggested the involvement of P_2U purinoceptors. The potentiation of Ca²⁺ rise was observed when acetylcholine and ATP were co-applied at submaximal concentrations (10 μM and 100 μM, respectively). We conclude that undifferentiated cells in the otocyst epithelium have CaCa²⁺ mobilizing systems activated by acetylcholine and ATP. © 1995 John Wiley & Sons, Inc.