ATP stimulates Ca2+-waves and gene expression in cultured human pulmonary fibroblasts

Given that extracellular ATP is markedly elevated in inflammation and is known to modulate fibroblast function, we examined the effects of exogenously added ATP on Ca²⁺-handling and gene expression in human pulmonary fibroblasts. Cells were loaded with the Ca²⁺-indicator dye fluo-4 and studied using confocal fluorimetry. Standard RT-PCR was used to probe gene expression. ATP (10^?5 M) evoked recurring Ca²⁺-waves which were completely occluded by cyclopiazonic acid (depletes the internal Ca²⁺-store) or the phospholipase inhibitor U73122. Pretreatment with ryanodine (10^?5 M), however, had no effect on the ATP-evoked responses. Regarding the receptor through which ATP acted, we found the ATP-response to be mimicked by UTP or ADP but not by adenosine or α,β-methylene-ATP, and to be blocked by the purinergic receptor blocker PPADS. The ATP-evoked response was greater and longer lasting within the nucleus than in the non-nuclear portion of the cytosol. RT-PCR showed that ATP also rapidly and dramatically increased gene expression of P2Y₄ receptors, the cytokine TGF-β (an important modulator of wound repair) and two matrix proteins (collagen A1 and fibronectin) approximately 4–5 times above baseline: this increase was not significantly affected by ryanodine but was abolished by PPADS. We conclude that, in human pulmonary fibroblasts, ATP acts upon P2Y receptors to liberate internal Ca²⁺ through ryanodine-insensitive channels, leading to a Ca²⁺-wave which courses throughout the cell and modulates gene expression.     

Differential Regulation of Ca<Superscript><Emphasis Type="Bold">2+</Emphasis></Superscript> Signaling and Membrane Trafficking by Multiple P2 Receptors in Brown Adipocytes

Extracellular ATP triggers changes in intracellular Ca²⁺, ion channel function, and membrane trafficking in adipocytes. The aim of the present study was to determine which P2 receptors might mediate the Ca²⁺ signaling and membrane trafficking responses to ATP in brown fat cells. RT-PCR was used to determine which P2 receptors are expressed in brown fat cells. Responses to nucleotide agonists and antagonists were characterized using fura-2 fluorescence imaging of Ca²⁺ responses, and FM 1-43 fluorescence imaging and membrane capacitance measurements to assess membrane trafficking. The pharmacology of the Ca²⁺ responses fits the properties of the P2Y receptors for which mRNA is expressed, but the agonist and antagonist sensitivity of the membrane-trafficking response was not consistent with any P2 receptor described to date. Brown adipocytes expressed mRNA for P2Y₂, P2Y₆, and P2Y₁₂ metabotropic receptors and P2X₁, P2X₂, P2X₃, P2X₄, P2X₅, and P2X₇ ionotropic receptors. The agonists ATP, ADP, UTP, UDP and 2′, 3′-(benzoylbenzoyl) ATP (BzATP) increased intracellular Ca²⁺, while 100 μM suramin, pyridoxal-phosphate-6-azophenyl-2′ 4′-disulfonic acid (PPADS), or Reactive Blue 2 partially blocked Ca²⁺ responses. ATP, but not ADP, UTP, UDP or BzATP activated membrane trafficking. The membrane response could be blocked completely with 1 μM PPADS but not by the antagonist MRS2179. We conclude that multiple P2 receptors mediate the ATP responses of brown fat cells, and that membrane trafficking is regulated by a P2 receptor showing unusual properties.     

ROS Production via P2Y1-PKC-NOX2 Is Triggered by Extracellular ATP after Electrical Stimulation of Skeletal Muscle Cells

During exercise, skeletal muscle produces reactive oxygen species (ROS) via NADPH oxidase (NOX2) while inducing cellular adaptations associated with contractile activity. The signals involved in this mechanism are still a matter of study. ATP is released from skeletal muscle during electrical stimulation and can autocrinely signal through purinergic receptors; we searched for an influence of this signal in ROS production. The aim of this work was to characterize ROS production induced by electrical stimulation and extracellular ATP. ROS production was measured using two alternative probes; chloromethyl-2,7- dichlorodihydrofluorescein diacetate or electroporation to express the hydrogen peroxide-sensitive protein Hyper. Electrical stimulation (ES) triggered a transient ROS increase in muscle fibers which was mimicked by extracellular ATP and was prevented by both carbenoxolone and suramin; antagonists of pannexin channel and purinergic receptors respectively. In addition, transient ROS increase was prevented by apyrase, an ecto-nucleotidase. MRS2365, a P2Y₁ receptor agonist, induced a large signal while UTPyS (P2Y₂ agonist) elicited a much smaller signal, similar to the one seen when using ATP plus MRS2179, an antagonist of P2Y₁. Protein kinase C (PKC) inhibitors also blocked ES-induced ROS production. Our results indicate that physiological levels of electrical stimulation induce ROS production in skeletal muscle cells through release of extracellular ATP and activation of P2Y1 receptors. Use of selective NOX2 and PKC inhibitors suggests that ROS production induced by ES or extracellular ATP is mediated by NOX2 activated by PKC.     

Vasoactive intestinal peptide reduces oxidative stress in pancreatic acinar cells through the inhibition of NADPH oxidase

Vasoactive intestinal peptide (VIP) attenuates experimental acute pancreatitis (AP) by inhibition of cytokine production from inflammatory cells. It has been suggested that reactive oxygen species (ROS) as well as cytokines play pivotal roles in the early pathophysiology of AP. This study aimed to clarify the effect of VIP on the oxidative condition in pancreas, especially pancreatic acinar cells (acini). Hydrogen peroxide (H₂O₂)-induced intracellular ROS, assessed with CM-H₂DCFDA, increased time- and dose-dependently in acini isolated from rats. Cell viability due to ROS-induced cellular damage, evaluated by MTS assay, was decreased with ≥100 μmol/L H₂O₂. VIP significantly inhibited ROS production from acini and increased cell viability in a dose-dependent manner. Expression of antioxidants including catalase, glutathione reductase, superoxide dismutase (SOD) 1 and glutathione peroxidase was not altered by VIP except for SOD2. Furthermore, Nox1 and Nox2, major components of NADPH oxidase, were expressed in pancreatic acini, and significantly increased after H₂O₂ treatment. Also, NADPH oxidase activity was provoked by H₂O₂. VIP decreased NADPH oxidase activity, which was abolished by PKA inhibitor H89. These results suggested that VIP affected the mechanism of ROS production including NADPH oxidase through induction of a cAMP/PKA pathway. In conclusion, VIP reduces oxidative stress in acini through the inhibition of NADPH oxidase. These results combined with findings of our previous study suggest that VIP exerts its protective effect in pancreatic damage, not only through an inhibition of cytokine production, but also through a reduction of the injury caused by oxidative stress.     

Inhibition of stomatal opening in sunflower leaves by carbon monoxide,and reversal of inhibition by light

When leaves of Helianthus annuus, whose stomates had been opened in the dark in the absence of CO₂, were exposed to 25% carbon monoxide (CO), stomatal conductivity for water vapor decreased from about 0.4 to 0.2 cm·s^-1. The CO effect on stomatal aperture required a CO/O₂ ratio of about 25. As this ratio was decreased the stomata opened, indicating that inhibitio of cytochrome-c oxidase by CO is competitive in respect to O₂. Photosynthetically active red light was unable to reverse CO-induced stomatal closure even at high irradiances, when CO₂ was absent. When it was present, stomatal opening was occasionally, but not consistently observed. Carbon monoxide did not inhibit photosynthetic carbon reduction in leaves of Helianthus.In contrast to red light, very weak blue light (405 nm) increased the stomatal aperture in the presence of CO. It also increased leaf ATP/ADP ratios which had been decreased in the presence of CO. The blue-light effect was not related to photosynthesis. Neither could it be explained by photodissociation of the cytochrome a ₃-CO complex which has an absorption maximum at 430 nm. The data indicate that ATP derived from mitochondrial oxidative phosphorylation provides energy for stomatal opening in sunflower leaves in the dark as well as in the light. Indirect transfer of ATP from chloroplasts to the cytosol via the triose phosphate/phosphoglycerate exchange which is mediated by the phosphate translocator of the chloroplast envelope can support stomatal opening only if metabolite concentrations are high enough for efficient shuttle transfer of ATP. Blue light causes stomatal opening in the presence of CO by stimulating ATP synthesis.     

NADPH oxidase is involved in H2O2-induced differentiation of human promyelocytic leukaemia HL-60 cells

The expression and activity of NADPH oxidase increase when HL‐60 cells are induced into terminally differentiated cells. However, the function of NADPH oxidase in differentiation is not well elucidated. With 150–500 μM H₂O₂ inducing differentiation of HL‐60 cells, we measured phagocytosis of latex beads and investigated cell electrophoresis. Two inhibitors of NADPH oxidase, DPI (diphenyleneiodonium) and APO (apocynin), blocked the differentiation potential of cells induced by 200 μM H₂O₂. However, H₂O₂ stimulated the generation of intracellular superoxide (O₂ ^{? ?}), which decreased in the presence of the two inhibitors. DPI also inhibited H₂O₂‐induced ERK (extracellular‐signal‐regulated kinase) activation, as detected by Western blotting. Furthermore, PD98059, the inhibitor of the ERK pathway, inhibited the differentiation of HL‐60 cells induced by H₂O₂. This shows that H₂O₂ can activate NADPH oxidase, leading to O₂ ^{? ?} production, followed by ERK activation and ultimately resulting in the differentiation of HL‐60 cells. The data indicate that NADPH oxidase is an important cell signal regulating cell differentiation.     

Insulin-induced NADPH oxidase activation promotes proliferation and matrix metalloproteinase activation in monocytes/macrophages

Insulin stimulates superoxide (O₂^?) production in monocytes and macrophages. However, the mechanisms through which insulin induces O₂^? production are not completely understood. In this study, we (a) characterized the enzyme and the pathways involved in insulin-stimulated O₂^? production in human monocytes and murine macrophages, and (b) analyzed the consequences of insulin-stimulated O₂^? production on the cellular phenotype in these cells. We showed that insulin stimulated O₂^? production, and promoted p47^phox translocation to the plasma membrane. Insulin-induced O₂^? production and p47^phox translocation were prevented in the presence of specific inhibitors of PI3K and PKC. Insulin-mediated NADPH oxidase activation stimulated MMP-9 activation in monocytes and cell proliferation in macrophages. The effect of insulin on these phenotypic responses was mediated through NFκB, p38MAPK, and ERK 1/2 activation. Small-interfering RNA-specific gene silencing targeted specifically against Nox2 reduced the cognate protein expression, decreased insulin-induced O₂^? production, inhibited the turn on of NFκB, p38MAPK, and ERK 1/2, and reduced cell proliferation in macrophages. These findings suggest a pivotal role for NADPH oxidase in insulin-induced proliferation and proteolytic activation in monocytes and macrophages, respectively, and identify a pathway that may play a pathological role in hyperinsulinemic states.     

ATP activates P2X receptors to mediate gap junctional coupling in the cochlea

ATP is an important extracellular signaling molecule and can activate both ionotropic (P2X) and metabotropic purinergic (P2Y) receptors to influence cellular function in many aspects. Gap junction is an intercellular channel and plays a critical role in hearing. Here, we report that stimulation of ATP reduced gap junctional coupling between cochlear supporting cells. This uncoupling effect could be evoked by nanomolar physiological levels of ATP. A P2X receptor agonist benzoylbenzoyl-ATP (BzATP) but not a P2Y receptor agonist UTP stimulated this uncoupling effect. Application of P2X receptor antagonists pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS, 50 μM) or oxidized ATP (oATP, 0.1 mM) eliminated this uncoupling effect. We further found that ATP activated P2X receptors in the cochlear supporting cells allowing Ca²⁺ influxing, thereby increasing intracellular Ca²⁺ concentration to mediate gap junctions. These data suggest that ATP can mediate cochlear gap junctions at the physiological level by the activation of P2X receptors rather than P2Y receptors. This P2X receptor-mediated purinergic control on the cochlear gap junctions may play an important role in the regulation of K⁺-recycling for ionic homeostasis in the cochlea and the reduction of hearing sensitivity under noise stress for protection.     

Extracellular ATP induces spikes in cytosolic free Ca but not in NADPH oxidase activity in neutrophils

In order to establish whether non-mitochondrial oxidase activity in human neutrophils is tightly related to cytosolic Ca²⁺ concentration, we simultaneously measured Ca²⁺ oscillations induced by ATP and oxidant production in single adherent neutrophils using confocal microscopy. ATP induced fast damped Ca²⁺ spikes with a period of 15 s and slower irregular spikes with a period greater than 50 s. Spikes in Ca²⁺ occurred in the absence of Ca²⁺ influx, but the amplitude was damped by inhibition of Ca²⁺ influx. Using the oxidation of hydroethidine as a cytosolic marker of oxidant production, we show that the generation of reactive oxygen species by neutrophils adherent to glass was accelerated by ATP. The step-up in NADPH oxidase activity followed the first elevation of cytosolic Ca²⁺ but, despite subsequent spikes in Ca²⁺ concentration, no oscillations in oxidase activity could be detected. ATP induced spikes in Ca²⁺ in a very reproducible way and we propose that the Ca²⁺ signal is an on-switch for oxidase activity, but the activity is apparently not directly correlated with spiking activity in cytosolic Ca²⁺.     

Studies on the Nature and Activation of O2−−-Forming NADPH Oxidase of Leukocytes.: II. Relationships Between Phosphorylation of a Component of the Enzyme and Oxidase Activity

The activation of O₂^?_?-formation by neutrophil NADPH oxidase is associated with phosphorylation of several membrane and cytosolic proteins. In the membranes a phosphoprotein of 32 kDa belonging to the NADPH oxidase-cytochrome b_-245 system (P. Bellavite et al., Free Rad. Res. Commun., 1, 11 (1985)) showed the highest relative increase of ³²Pi incorporation. Concomitant with the phosphorylation, a shift of the apparent molecular mass of the protein from 31 to 32 kDa occurred. The time-course, the sensitivity to trifluoperazine and the dose-dependence of phosphorylation were similar to those of O₂^?_? forming activity, except that the latter showed a longer lag-time than the former. The increase of the 32kDa phosphoprotein was also comparable to the kinetics of cytochrome b_-245 reduction by anaerobically activated neutrophils. The phosphorylation and the NADPH oxidase were triggered by various stimulants including phorbol myristate acetate, opsonized zymosan, arachidonic acid and sodium fluoride. With arachidonic acid the O₂^?_? formation was highly active but the phosphorylation was low. With fluoride the enzyme activity was reversible upon removal of the stimulant but the phosphorylation of the 32 kDa peptide was not reversible. Neutrophils treated with PMA at 17°C showed phosphorylation but not activation. The results indicate that phosphorylation of a component of NADPH oxidase is a fundamental but probably not sufficient event in the activation mechanism of the enzyme.     

Cytochrome oxidase from Pseudomonas aeruginosa. III. Reduction of hydroxylamine

Pseudomonas aeruginosa cytochrome oxidase, which reduces nitrite and oxygen, is also capable of reducing hydroxylamine to ammonia.The K_m for hydroxylamine reduction is 6 · 10^?4M compared to 5 · 10^?5M for nitrite reduction. NADH, NADPH, reduced P. aeruginosa cytochrome c₅₅₁, and reduced P. aeruginosa copper protein were ineffective as electron donors for hydroxylamine reduction whereas reduced pyocyanine and methylene blue acted as electron mediators.Hydroxylamine reduction did not require the presence of Mn²⁺ of FAD and was not inhibited by prolonged dialysis versus sodium diethyldithiocarbamate. Cyanide, nitrite, and CO were very effective inhibitors.Removal of heme d and its reconstitution, as well as inhibition by CO, suggest that the reduction of hydroxylamine, like the reduction of nitrite or oxygen, proceeds via the heme d.     

P2X7 Receptors in Rat Brain: Presence in Synaptic Terminals and Granule Cells

ATP stimulates [Ca²⁺]_i increases in midbrain synaptosomes via specific ionotropic receptors (P2X receptors). Previous studies have demonstrated the implication of P2X₃ subunits in these responses, but additional P2X subunits must be involved. In the present study, ATP and BzATP proved to be able to induce intrasynaptosomal calcium transients in the midbrain synaptosomes, their effects being potentiated when assayed in a Mg₂₊-free medium. Indeed, BzATP was shown to be more potent than ATP, and their effects could be inhibited by PPADS and KN-62, but not by suramin. This activity profile is consistent with the presence of functional P2X₇ receptors in the midbrain terminals. The existence of presynaptic responses to selective P2X7 agonists could be confirmed by means of a microfluorimetric technique allowing [Ca²⁺]_i measurements in single synaptic terminals. Additionally, the P2X₇ receptor protein could be identified in the midbrain synaptosomes and in axodendritic prolongations of cerebellar granule cells by immunochemical staining.     

The role of calcitonin gene-related peptide (CGRP) in macrophages: the presence of functional receptors and effects on proliferation and differentiation into osteoclast-like cells

It has been shown that both calcitonin gene-related peptide (CGRP) and amylin bind weakly to calcitonin (CT) receptors in osteoclast-like cells formed in vitro and inhibit bone resorption by a cAMP-dependent mechanism. Osteoclasts are thought to be derived from cells of the monocyte macrophage lineage, in which CGRP, but not CT, induces cAMP production. In this study, we determined the presence of functional receptors for CGRP in mouse alveolar macrophages and the effects of this peptide on proliferation and osteoclastic differentiation in mouse alveolar and bone marrow-derived macrophages. Human CT did not stimulate cAMP production in macrophages. Human CGRP stimulated cAMP production in mouse alveolar macrophages and bone marrow-derived macrophages dose-dependently. Human amylin, which has 43% homology with human CGRP, also stimulated these macrophages to produce cAMP, but only at a 100-fold higher concentration. The increment in cAMP production induced by human CGRP and amylin was abolished by the addition of human CGRP(8–37), a selective antagonist for CGRP receptors. Specific binding of [¹²⁵I]human CGRP to alveolar macrophages was detected (dissociation constant, 2.5 × 10⁻⁸ M; binding sites, 1.4 × 10⁴/cell). Amylin, but not CT, displaced the bound [¹²⁵I]human CGRP from alveolar macrophages, but at a 100-fold higher concentration. No specific binding of [¹²⁵I]human CT and [¹²⁵I]human amylin to alveolar macrophages could be detected. Pretreatment with human CGRP for 24 h dose-dependently suppressed DNA synthesis in alveolar macrophages induced by granulocyte-macrophage colony-stimulating factor (GM-CSF). CGRP also suppressed the number of macrophage colonies formed from bone marrow cells induced by macrophage colony-stimulating factor (M-CSF). Pre-treatment of alveolar macrophages with CGRP inhibited differentiation into osteoclast-like cells in co-cultures with primary osteoblastic cells in the presence of 1α,25-dihydroxy vitamin D₃. These results indicate that specific receptors for CGRP are present in macrophages and that CGRP modulates proliferation and differentiation of macrophages into osteoclast-like cells by a receptor-mediated mechanism involving cAMP.     

Vascular smooth muscle cells from small human omental arteries express P2X1 and P2X4 receptor subunits

Stimulation of P2X receptors by ATP in vascular smooth muscle cells (VSMCs) is proposed to mediate vascular tone. However, understanding of P2X receptor-mediated actions in human blood vessels is limited, and therefore, the current work investigates the role of P2X receptors in freshly isolated small human gastro-omental arteries (HGOAs). Expression of P2X1 and P2X4 receptor subunit messenger RNA (mRNA) and protein was identified in individual HGOA VSMCs using RT-PCR and immunofluorescent analysis and using Western blot in multi-cellular preparations. ATP of 10 μmol/l and αβ-meATP of 10 μmol/l, a selective P2X receptor agonist, evoked robust increases in [Ca²⁺]_i in fluo-3-loaded HGOA VSMCs. Pre-incubation with 1 μmol/l NF279, a selective P2X receptor antagonist, reduced the amplitude of αβ-meATP-induced increase in [Ca²⁺]_i by about 70 %. ATP of 10 μmol/l and αβ-meATP of 10 μmol/l produced similar contractile responses in segments of HGOA, and these contractions were greatly reduced by 2 μmol/l NF449, a selective P2X receptor inhibitor. These data suggest that VSMCs from HGOA express P2X1 and P2X4 receptor subunits with homomeric P2X1 receptors likely serving as the predominant target for extracellular ATP.

Electronic supplementary material

The online version of this article (doi:10.1007/s11302-014-9415-6) contains supplementary material, which is available to authorized users.     

Multiple P2Y receptors couple to calcium-dependent,chloride channels in smooth muscle cells of the rat pulmonary artery

Background

Uridine 5''-triphosphate (UTP) and uridine 5''-diphosphate (UDP) act via P2Y receptors to evoke contraction of rat pulmonary arteries, whilst adenosine 5''-triphosphate (ATP) acts via P2X and P2Y receptors. Pharmacological characterisation of these receptors in intact arteries is complicated by release and extracellular metabolism of nucleotides, so the aim of this study was to characterise the P2Y receptors under conditions that minimise these problems.

Methods

The perforated-patch clamp technique was used to record the Ca²⁺-dependent, Cl^- current (I_Cl,Ca) activated by P2Y receptor agonists in acutely dissociated smooth muscle cells of rat small (SPA) and large (LPA) intrapulmonary arteries, held at -50 mV. Contractions to ATP were measured in isolated muscle rings. Data were compared by Student''s t test or one way ANOVA.

Results

ATP, UTP and UDP (10^-4M) evoked oscillating, inward currents (peak = 13–727 pA) in 71–93% of cells. The first current was usually the largest and in the SPA the response to ATP was significantly greater than those to UTP or UDP (P < 0.05). Subsequent currents tended to decrease in amplitude, with a variable time-course, to a level that was significantly smaller for ATP (P < 0.05), UTP (P < 0.001) and UDP (P < 0.05) in the SPA. The frequency of oscillations was similar for each agonist (mean≈6–11.min^-1) and changed little during agonist application. The non-selective P2 receptor antagonist suramin (10^-4M) abolished currents evoked by ATP in SPA (n = 4) and LPA (n = 4), but pyridoxalphosphate-6-azophenyl-2'',4''-disulphonic acid (PPADS) (10^-4M), also a non-selective P2 antagonist, had no effect (n = 4, 5 respectively). Currents elicited by UTP (n = 37) or UDP (n = 14) were unaffected by either antagonist. Contractions of SPA evoked by ATP were partially inhibited by PPADS (n = 4) and abolished by suramin (n = 5). Both antagonists abolished the contractions in LPA.

Conclusion

At least two P2Y subtypes couple to I_Cl,Ca in smooth muscle cells of rat SPA and LPA, with no apparent regional variation in their distribution. The suramin-sensitive, PPADS-resistant site activated by ATP most resembles the P2Y₁₁ receptor. However, the suramin- and PPADS-insensitive receptor activated by UTP and UDP does not correspond to any of the known P2Y subtypes. These receptors likely play a significant role in nucleotide-induced vasoconstriction.     

Interaction of vasoactive intestinal peptide (VIP) with a mouse adrenal cell line (Y-1): specific binding and biological effects.

Y-1 cells specifically bind radiolabelled vasoactive intestinal peptide (VIP) with a dissociation constant of about 10^?9 M. [¹²⁵I]-VIP bound was not displaced by ACTH. VIP stimulates both steroid and cAMP production, with half-maximal stimulation at 10^?9 and 10^?8 M, respectively. At maximal concentration VIP produces the same stimulation of steroidogenesis as ACTH, but induced three times lower production of cAMP than ACTH. Y-1 DNA synthesis is inhibited by VIP in a dose-dependent manner with half-maximal inhibition at 10^?8 M. At submaximal concentrations the effects of VIP and ACTH on cAMP and steroid production and on inhibition of DNA synthesis are additive. Similar additive effects on cAMP production and on inhibition of DNA synthesis were observed at submaximal ACTH and maximal VIP concentration, but the phenomenon was no longer seen at maximal concentrations of both peptides. These data suggest that in Y-1 cells VIP stimulates, through its own distinct receptors, only a part of the pool of adenylate cyclase sensitive to ACTH.     

P2-purinergic receptors are coupled to two signal transduction systems leading to inhibition of cAMP generation and to production of inositol trisphosphate in rat hepatocytes

Stimulation of P2-purinergic receptors by ATP resulted in activation of phosphorylase, which was associated with marked production of inositol trisphosphate (Ins-P3), in rat hepatocytes. ATP also inhibited forskolin-induced accumulation of cAMP in the presence of a phosphodiesterase inhibitor. On the contrary, adenosine or AMP never inhibited the cAMP accumulation, but increased hepatocyte cAMP; the stimulation was antagonized by a methylxanthine. Thus, P1-purinergic receptors are linked to adenylate cyclase in a stimulatory fashion in hepatocytes. Various kinds of purine nucleotides stimulating P2-receptors can be divided into two groups on the basis of their relative abilities to stimulate Ins-P3 production and to inhibit cAMP accumulation; the first group including adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), ADP, 5-adenylyl imidodiphosphate, GTP, and guanosine 5'-O-(3-thiotriphosphate) has an efficacy similar to that of ATP, and the second group of nucleotides including alpha, beta-methyleneadenosine 5'-triphosphate, beta, gamma-methyleneadenosine 5'-triphosphate (App(CH)2)p), and GDP exerts considerable inhibitory effects on cAMP accumulation, but only slight effects on inositol lipid metabolism. Treatment of hepatocytes with islet-activating protein, pertussis toxin, blocked the nucleotide-induced inhibition of cAMP accumulation, but exerted only a small effect on Ins-P3 production. In membranes prepared from hepatocytes, forskolin-stimulated adenylate cyclase was inhibited by GTP. This GTP-induced inhibition of the enzyme was susceptible to islet-activating protein and dependent on the concentration of ATP (or its derivatives, ATP gamma S or App(CH2)p). It is concluded that there are two types of P2-purinergic receptors: one is linked to adenylate cyclase via an inhibitory guanine nucleotide regulatory protein (Gi) and the other is linked to phospholipase C.     

The effects of extracellular nucleotides on [Ca2+]i signalling in a human‐derived renal proximal tubular cell line (HKC‐8)

HKC‐8 cells are a human‐derived renal proximal tubular cell line and provide a useful model system for the study of human renal cell function. In this study, we aimed to determine [Ca²⁺]_i signalling mediated by P2 receptor in HKC‐8. Fura‐2 and a ratio imaging method were employed to measure [Ca²⁺]_i in HKC‐8 cells. Our results showed that activation of P2Y receptors by ATP induced a rise in [Ca²⁺]_i that was dependent on an intracellular source of Ca²⁺, while prolonged activation of P2Y receptors induced a rise in [Ca²⁺]_i that was dependent on intra‐ and extracellular sources of Ca²⁺. Pharmacological and molecular data in this study suggests that TRPC4 channels mediate Ca²⁺ entry in coupling to activation of P2Y in HKC‐8 cells. U73221, an inhibitor of PI‐PLC, did not inhibit the initial ATP‐induced response; whereas D609, an inhibitor of PC‐PLC, caused a significant decrease in the initial ATP‐induced response, suggesting that P2Y receptors are coupled to PC‐PLC. Although P2X were present in HKC‐8, The P2X agonist, α,β me‐ATP, failed to cause a rise in [Ca²⁺]_i. However, PPADS at a concentration of 100 µM inhibits the ATP‐induced rise in [Ca²⁺]_i. Our results indicate the presence of functional P2Y receptors in HKC‐8 cells. ATP‐induced [Ca²⁺]_i elevation via P2Y is tightly associated with PC‐PLC and TRP channel. J. Cell. Biochem. 109: 132–139, 2010. © 2009 Wiley‐Liss, Inc.     

Comparative pharmacology of two D1-like dopamine receptors cloned from the silkworm Bombyx mori

Dopamine (DA) is a physiologically important biogenic amine in insect peripheral and nervous tissues. We recently cloned two DA receptors (BmDopR1 and BmDopR2) from the silkworm Bombyx mori and identified them as D1-like receptors, which activate adenylate cyclase to increase intracellular cAMP levels. In this study, these two receptors were stably expressed in HEK-293 cells, and the dose-responsiveness to DA and their pharmacological properties were examined using cAMP assays. BmDopR1 showed a dose-dependent increase in cAMP levels at DA concentrations up to 10^?7 M with EC₅₀ of 3.30 nM, while BmDopR2 required 10^?6 M DA for activation. In BmDopR1-expressing cells, DA at 10^?6–10^?4 M induced 30–50% lower cAMP production than 10^?7 M DA. BmDopR2-expressing cells showed a standard sigmoidal dose–response, with maximum cAMP levels attained with 10^?5–10^?4 M DA and EC₅₀ of 1.30 μM. Both receptors had similar agonist profiles, and the typical vertebrate D1-like receptor agonist SKF-38393 was ineffective. Experiments with antagonists revealed that BmDopR1 exhibits D1-like features. However, the pharmacology of BmDopR2 was distinct from D1-like receptors; the typical vertebrate D1-like receptor antagonist SCH-23390 was less potent than the nonselective antagonist flupenthixol and the D2-like receptor antagonist chlorpromazine. The rank order of activities of several antagonists for BmDopR1 and BmDopR2 was more similar to that of Drosophila melanogaster DA receptors than Apis mellifera DA receptors. These data suggest that DA receptors could be potential targets for specific insecticides or insectistatics.     

Mechanisms for Facilitation of Nitric Oxide-Evoked [3H]GABA Release by Removal of Hydroxyl Radical

Abstract: We have investigated the mechanisms for enhancement of nitric oxide (NO)-evoked γ-[³H]aminobutyric acid ([³H]GABA) release from mouse cerebro-cortical neurons by hydroxyl radical (^?OH) scavengers. ^?OH scavengers, such as N,N′-dimethylthiourea (DMTU), uric acid, and mannitol, dose-dependently facilitated NO-evoked [³H]GABA release evoked by NO liberated from S-nitroso-N-acetylpenicillamine. Ionomycin-evoked [³H]GABA release, which was significantly inhibited by hemoglobin and an NO synthase, N^G-methyl-l -arginine, was also enhanced by DMTU. These results indicate that GABA release evoked by both endogenous and exogenous NO is facilitated by ^?OH scavengers. These enhancing actions of ^?OH scavengers were completely abolished by Ca²⁺ removal from incubation buffer and by an l -type voltage-dependent Ca²⁺ channel (VDCC) inhibitor, nifedipine, whereas each ^?OH scavenger showed no effects on [³H]GABA release in the absence of NO. Inhibitors for P/Q- and N-type VDCCs had no effects on the enhancement. NO-induced ⁴⁵Ca²⁺ influx was also dose-dependently enhanced by ^?OH scavengers, although ⁴⁵Ca²⁺ influx was not altered by ^?OH scavengers in the absence of NO. Nifedipine abolished this enhancement of the NO-induced ⁴⁵Ca²⁺ influx by ^?OH scavengers. These results indicate that the removal of ^?OH by its scavengers facilitates the NO-evoked [³H]GABA release dependent on Ca²⁺ and that this enhancement is due to the increase in Ca²⁺ influx via L-type VDCCs.