Biomedical subjects

Y Uezono

Publications and source records attributed to Y Uezono.

At least 19 recordsLinked to original sources

PubMed2003

Mechanisms of cytosolic Ca2+ suppression by prostaglandin E2 receptors in rat melanotrophs.

We have previously reported that voltage-dependent Ca2+ (VDC) channels of rat melanotrophs are inhibited by prostaglandin E2 (PGE2). In this study, mechanisms involved in the inhibitory actions of PGE2 receptors of rat melanotrophs were analysed using reverse transcriptase-polymerase chain reaction (RT-PCR), Ca2+-imaging and whole-cell, patch-clamp techniques with recently developed EP agonists, each of which is selective for the known four subclasses of EP receptors (EP1-4). PGE2 reversibly suppressed the cytosolic Ca2+ concentration ([Ca2+]i). The maximum reduction in [Ca2+]i by PGE2 was comparable to that by dopamine or to that by extracellular Ca2+ removal. RT-PCR analysis of all four EP receptors revealed that EP3 and EP4 receptor mRNAs were expressed in the intermediate lobe. The effects of PGE2 to suppress [Ca2+]i were mimicked by the selective EP3 agonist, ONO-AE-248, whereas three other EP agonists, ONO-DI-004 (EP1), ONO-AE1-259 (EP2) and ONO-AE1-329 (EP4), had little or no effect on [Ca2+]i. All four G-protein activated inward rectifying K+ (GIRK) channel mRNAs were identified in intermediate lobe tissues by RT-PCR. Dopamine concentration-dependently activated GIRK currents, whereas PGE2 did not activate GIRK currents, even at the concentration causing maximal inhibition of VDC channels. These results suggest that PGE2 acts on EP3 receptors to suppress Ca2+ entry of rat melanotrophs by selectively inhibiting VDC channels of these cells. We have compared the possible cellular and molecular mechanisms of inhibition by dopamine and PGE2.

Animals↗

PubMed2001

Production of cAMP by adrenomedullin in human oligodendroglial cell line KG1C: comparison with calcitonin gene-related peptide and amylin.

The actions and the presence of adrenomedullin (AM) were investigated in cultured human oligodendroglial cell line KG1C. AM and AM mRNA were detected in KG1C cells by immunohistochemistry and RT-PCR. mRNAs for calcitonin receptor-like receptor (CRLR) and receptor-activity-modifying proteins (RAMPs) 1, 2 and 3 but not for calcitonin receptors were detected in the cells, while mRNAs for CRLR, calcitonin receptors and all RAMPs were detected in the human cerebellum. Application of AM resulted in time- and concentration-dependent increases in the cAMP level of KG1C cells. Calcitonin gene-related peptide (CGRP) and amylin, peptides structurally related to AM, also increased cAMP. The potencies for the cAMP production of the three peptides were CGRP > or =AM >> amylin with EC(50) of 8, 18, 90 nM, respectively. The responses induced by AM were strongly inhibited by the CGRP(1) receptor antagonist human CGRP(8-37), and inhibited also by the AM receptor antagonist human AM(22-52). In contrast, the responses induced by CGRP or amylin were inhibited only by CGRP(8-37) and not by AM(22-52). The responses induced by all three peptides were unaffected by the amylin receptor antagonist human amylin(8-37). The CGRP(2) receptor agonist human [Cys(Acm)(2,7)]CGRP significantly increased the cAMP level but the increase was smaller than that caused by CGRP. This increase in cAMP was unaffected by CGRP(8-37), AM(22-52) or by amylin(8-37). These results suggest that in KG1C cells, AM increases cAMP through AM and CGRP(1) receptors, whereas CGRP does so through CGRP(1) and CGRP(2) receptors, and amylin exerts its effects through CGRP(1) receptors. Collectively, these findings imply that AM released from oligodendroglial cells may play a role in the regulation of oligodendrocytes via autocrine/paracrine through AM receptors and CGRP(1) receptors.

Adrenal Medulla↗

PubMed2001

Down-regulation of cell surface insulin receptors by sarco(endo)plasmic reticulum Ca2+-ATPase inhibitor in adrenal chromaffin cells.

Long-term (> or =12 h) treatment of cultured bovine adrenal chromaffin cells with thapsigargin (TG), an inhibitor of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), caused a time (t(1/2)=16.3 h)- and concentration (IC50=37.8 nM)-dependent decrease of cell surface 125I-insulin binding by 35%, but did not change the Kd value. TG caused a sustained increase of cytoplasmic concentration of Ca2+ ([Ca2+]c) in a biphasic manner, and the effect of TG on 125I-insulin binding was abolished by BAPTA-AM. Western blot analysis showed that TG lowered insulin receptor (IR) beta-subunit level in membrane, but did not alter total cellular levels of IR precursor and IR beta-subunit. Internalization of cell surface IR, as measured by using brefeldin A, an inhibitor of vesicular exit from the trans-Golgi network (TGN), was not changed by TG. These results suggest that inhibition of SERCA by TG and the subsequent increase of [Ca2+]c down-regulates cell surface IR by retarding externalization of IR from the TGN.

Adrenal Glands↗

PubMed2001

Aquaporin subtypes in rat cerebral microvessels.

We investigated the expression of aquaporin (AQP) subtypes in the rat cerebral microvessels by reverse transcription-polymerase chain reaction, immunoblotting and immunohistochemistry. mRNA for AQP4, but not for AQP1, 2, 3 or 5, was detected in the microvessels. Immunoblot analysis showed that AQP4 protein was detected as a 30 kDa band with higher molecular weight bands. Immunohistochemical staining showed that AQP4 was located on cell surface of the cerebral microvessels. These results suggest that AQP4 in the cerebral microvessels is involved in the regulation of water transport between blood and brain.

Animals↗

PubMed2001

Sequential changes in transforming growth factor (TGF)-beta1 concentration in synovial fluid and mRNA expression of TGF-beta1 receptors in chondrocytes after immobilization of rabbit knees.

We have previously reported that a combination of transforming growth factor (TGF)-beta1 and basic fibroblast growth factor (bFGF) synergistically increases the proliferation of chondrocytes obtained from knee joint immobilized for 7-14 days in male Japanese white rabbits. In the present study, we performed experiments with chondrocytes and syn ovial fluid obtained from rabbit knees immobilized for 0-42 days, to clarify the sequential changes in TGF-beta1 and bFGF concentrations in synovial fluid and the mRNA expression of TGF-beta1 receptor type I (RI) and II (RII) in chondrocytes after immobilization. The combination of TGF-beta1 and bFGF had a synergistic effect on the proliferation of chondrocytes obtained from knee joints immobilized for 7-14 days. The concentration of TGF-beta1 in synovial fluid was significantly higher (up to 3.6-fold) at 7-28 days after immobilization compared with that at 2 days. The mRNA for RI and RII was expressed during the whole immobilization period. The con centration of bFGF was kept at the same level at 2-7 days after immobilization, and gradually decreased thereafter. In the early stages of degenerated cartilage, up to 14 days after immobilization, the concentrations of both TGF-beta1 and bFGF were higher in the synovial fluid and mRNA expression of TGF-beta1 receptors in chondrocytes was kept.

Activin Receptors, Type I↗

PubMed2001

Selective expression of aquaporin 1, 4 and 5 in the rat middle ear.

The middle ear cavity is an air-filled space that must be maintained for effective sound transmission to the inner ear. To examine the mechanisms of water homeostasis in the middle ear, we investigated whether aquaporins (AQPs), a family of water-permeable channels, were expressed in the middle ear. Reverse transcription-polymerase chain reaction and immunoblot analyses revealed that mRNAs encoding AQP1, 4 and 5 (but not 2 or 3) subtypes were expressed in rat middle ear epithelium; AQP1, 4 and 5 were detected as 28-, 30- and 30-kDa proteins, respectively. Immunohistochemical analysis showed that AQP1 was localized at capillary endothelial cells and fibroblasts in lamina propria mucosae; AQP4 was present solely at the basolateral membrane of ciliated cells, whereas AQP5 was on the apical surface of ciliated cells as well as of flat and columnar epithelial cells. The characteristic different localizations of AQP1, 4 and 5 subtypes in the middle ear suggest that middle ear water homeostasis requires the coordinated operation of these AQPs.

Animals↗

PubMed2001

Heterogeneous increases of cytoplasmic calcium: distinct effects on down-regulation of cell surface sodium channels and sodium channel subunit mRNA levels.

1. Long-term (> or = 12 h) treatment of cultured bovine adrenal chromaffin cells with A23187 (a Ca(2+) ionophore) or thapsigargin (TG) [an inhibitor of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)] caused a time- and concentration-dependent reduction of cell surface [(3)H]-saxitoxin (STX) binding capacity, but did not change the K:(D:) value. In A23187- or TG-treated cells, veratridine-induced (22)Na(+) influx was reduced (with no change in veratridine EC(50) value) while it was enhanced by alpha-scorpion venom, beta-scorpion venom, or Ptychodiscus brevis toxin-3, like in nontreated cells. 2. The A23187- or TG-induced decrease of [(3)H]-STX binding was diminished by BAPTA-AM. EGTA also inhibited the decreasing effect of A23187. A23187 caused a rapid, monophasic and persistent increase in intracellular concentration of Ca(2+) ([Ca(2+)](i)) to a greater extent than that observed with TG. 2,5-Di-(t-butyl)-1,4-benzohydroquinone (DBHQ) (an inhibitor of SERCA) produced only a rapid monophasic increase in [Ca(2+)](i), without any effect on [(3)H]-STX binding. 3. Reduction in [(3)H]-STX binding capacity induced by A23187 or TG was attenuated by Gö6976 (an inhibitor of conventional protein kinase C) or calpastatin peptide (an inhibitor of calpain). When the internalization rate of cell surface Na(+) channels was measured in the presence of brefeldin A (an inhibitor of vesicular exit from the trans-Golgi network), A23187 or TG accelerated the reduction of [(3)H]-STX binding capacity. 4. Six hours treatment with A23187 lowered Na(+) channel alpha- and beta(1)-subunit mRNA levels, whereas TG had no effect. 5. These results suggest that elevation of [Ca(2+)](i) caused by A23187, TG or DBHQ exerted differential effects on down-regulation of cell surface functional Na(+) channels and Na(+) channel subunit mRNA levels.

Animals↗

PubMed2001

Adrenomedullin inhibits the pressor effects and decrease in renal blood flow induced by norepinephrine or angiotensin II in anesthetized rats.

Adrenomedullin (AM), a hypotensive peptide originally isolated from human pheochromocytoma, has been reported to regulate renal functions. In patients with glomerulonephritis, the serum levels of AM are elevated as well as hypertensive agents norepinephrine (NE) and angiotensin II (AII). The effects of AM on the NE- or AII-induced pressor effects and renal blood flow responses, however, are not well clarified. We examined the effects of AM on blood pressure and renal blood flow induced by NE or AII in anesthetized rats. Arterial blood pressure and renal blood flow were measured using a calibrated pressure transducer and a laser Doppler flowmeter, respectively. Drugs were injected into the tail vein with a syringe. Intravenous administration of AM (1-3 nmol/kg) decreased the arterial blood pressure in anesthetized rats in a dose-dependent manner, whereas it did not affect the renal blood flow. NE or AII administration in anesthetized rats caused both increases in blood pressure and decreases in renal blood flow. Simultaneous administration of AM with NE or All prevented the increasing effects of blood pressure and inhibited the decreases in renal blood flow caused by NE or AII. These findings suggest that AM may have a protective role against the pressor effects and decrease in renal blood flow caused by NE or AII.

Adrenomedullin↗

PubMed2001

GTP gamma S-induced Ca2+ activated Cl- currents: its stable induction by Gq alpha overexpression in Xenopus oocytes.

In native Xenopus oocytes, injection of guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) (30 mM, 5 nl) did not induce Cl- current in 11 out of 22 oocytes. Injection of increased concentration of GTPgammaS (100 mM, 5 nl) into the oocytes induced Cl- currents in 16 out of 17 oocytes; however, the size of the induced currents was extremely varied. In oocytes overexpressing Gq alpha, GTPgammaS (30 mM, 5 nl) faithfully evoked Ca2+-activated Cl- currents. These results indicate that heterogeneous expression of Gq alpha in Xenopus oocytes provides a useful system for studying the functional roles of Gq alpha in regulating cellular events.

Animals↗

PubMed2001

Up-regulation of cell surface sodium channels by cyclosporin A, FK506, and rapamycin in adrenal chromaffin cells.

Treatment of cultured bovine adrenal chromaffin cells with cyclosporin A (CsA) increased cell surface [(3)H]saxitoxin ([(3)H]STX) binding by 56% in a time (t(1/2) = 15.2 h)- and concentration (EC(50) = 2.9 microM)-dependent manner but did not change the K(d) value. In CsA-treated cells, veratridine-induced (22)Na(+) influx was augmented with no change in the EC(50) of veratridine; also, alpha- and beta-scorpion venom and Ptychodiscus brevis toxin-3 enhanced veratridine-induced (22)Na(+) influx in a more than additive manner, as in nontreated cells. CsA treatment for 1 to 24 h inhibited calcineurin activity, measured by the in vitro assay, with the IC(50) of 0.6 microM but did not alter cellular level of calcineurin. FK506 or rapamycin elevated [(3)H]STX binding by 36 or 25%, whereas GPI-1046, an immunophilin ligand incapable to inhibit calcineurin, or okadaic acid, an inhibitor of protein phosphatases 1 and 2A, had no increasing effect. The rise of [(3)H]STX binding by CsA was attenuated by the coincident treatment with brefeldin A (BFA), an inhibitor of vesicular exit from the trans-Golgi network. The internalization rate of cell surface Na(+) channels, as determined in the presence of BFA, was decreased in CsA (but not rapamycin)-treated cells (t(1/2) = 20.3 h), compared with nontreated cells (t(1/2) = 13.7 h). CsA treatment, however, did not elevate cellular levels of Na(+) channel alpha-subunit and Na(+) channel alpha- and beta(1)-subunit mRNAs. In CsA-treated cells, veratridine-induced (45)Ca(2+) influx via voltage-dependent Ca(2+) channels and catecholamine secretion were enhanced, whereas high K(+)-induced (45)Ca(+) influx was not. Thus, the inhibition of calcineurin or rapamycin-binding protein causes up-regulation of cell surface functional Na(+) channels via modulating externalization and internalization of Na(+) channels, thus enhancing Ca(2+) channel gating and catecholamine secretion.

Adrenal Glands↗

PubMed2001

Inhibition by tramadol of muscarinic receptor-induced responses in cultured adrenal medullary cells and in Xenopus laevis oocytes expressing cloned M1 receptors.

Tramadol is a widely used, centrally acting analgesic, but its mechanisms of action are not completely understood. Muscarinic receptors are known to be involved in neuronal function in the brain and autonomic nervous system, and much attention has been paid to these receptors as targets of analgesic drugs in the central nervous system. This study investigated the effects of tramadol on muscarinic receptors by using two different systems, i.e., a Xenopus laevis oocyte expression system and cultured bovine adrenal medullary cells. Tramadol (10 nM-100 microM) inhibited acetylcholine-induced currents in oocytes expressing the M1 receptor. Although GF109203X, a protein kinase C inhibitor, increased the basal current, it had little effect on the inhibition of acetylcholine-induced currents by tramadol. On the other hand, tramadol did not inhibit the current induced by AlF4-, a direct activator of GTP-binding protein. In cultured bovine adrenal medullary cells, tramadol (100 nM-100 microM) suppressed muscarine-induced cyclic GMP accumulation. Moreover, tramadol inhibited the specific binding of [3H]quinuclidinyl benzilate (QNB). Scatchard analysis showed that tramadol increases the apparent dissociation constant (Kd) value without changing the maximal binding (Bmax), indicating competitive inhibition. These findings suggest that tramadol at clinically relevant concentrations inhibits muscarinic receptor function via QNB-binding sites. This may explain the neuronal function and anticholinergic effect of tramadol.

Acetylcholine↗

PubMed2000

Post-translational reduction of cell surface expression of insulin receptors by cyclosporin A, FK506 and rapamycin in bovine adrenal chromaffin cells.

Long-term (>/=3 h) treatment of cultured bovine adrenal chromaffin cells with cyclosporin A (CsA) decreased cell surface (125)I-insulin binding by 62% in a concentration (IC(50)=18 microM)- and time (t(1/2)=16 h)-dependent manner, but did not change the K(d) value. FK506 (1 microM) or rapamycin (3 microM) treatment reduced (125)I-insulin binding. Western blot analysis showed that CsA treatment decreased insulin receptor (IR) beta-subunit level (t(1/2)=15 h) in membrane fraction, but did not alter total cellular levels of IR precursor and IR beta-subunit. Internalization rate of cell surface IR measured by using brefeldin A, an inhibitor of vesicular exit from the trans-Golgi network, was comparable between non-treated and CsA-treated cells. Thus, CsA, FK506 and rapamycin inhibit peptidyl prolyl cis-trans isomerase activities of cyclophilin and FK506-binding protein, and down-regulate IR presumably by reducing cell surface externalization of IR.

Animals↗

PubMed2000

Adrenomedullin receptors in rat cerebral microvessels.

To characterize the sites of action of adrenomedullin (AM) in the cerebral microvasculature, we studied the effect of AM on cyclic AMP (cAMP) level as well as expression of AM and its receptor in the rat cerebral microvessels. The microvessels were prepared from rat cerebral cortex by albumin flotation and glass bead filtration technique. AM and calcitonin gene-related peptide (CGRP) increased cAMP level in the microvessels in a concentration-dependent manner. The effect of AM was more than 100 times more potent than that of CGRP. The accumulation of cAMP by AM was inhibited by AM[22-52], an AM receptor antagonist, but not by CGRP[8-37], a CGRP receptor antagonist, suggesting that AM increased cAMP accumulation by acting on receptors specific to AM. [125I]AM binding to the microvessels was displaced by AM and less potently by AM[22-52]. The displacing potencies of CGRP and CGRP[8-37] were very weak. mRNAs for AM as well as calcitonin-receptor-like receptor and receptor-activity-modifying protein 2 which form a receptor specific to AM, were highly expressed in the microvessels. These results provide biochemical and pharmacological evidence that AM is produced in and acts on the cerebral microvessels in an autocrine/paracrine manner and is involved in regulation of cerebral microcirculation.

Adrenomedullin↗

PubMed2000

Characterization and functional role of leptin receptor in bovine adrenal medullary cells.

We report here the characterization and functional roles of the leptin receptor (ObR) in bovine adrenal medullary cells. The plasma membranes isolated from bovine adrenal medulla showed a single class of specific binding sites of (125)I-leptin with an apparent K(d) of 6.6 nM and B(max) of 62 fmol/mg protein. ObRa but not ObRb mRNA was detected in bovine adrenal medulla by reverse transcriptase-polymerase chain reaction. Incubation of cultured adrenal medullary cells with leptin (3-30 nM) for 20 min resulted in a significant increase in [(14)C]catecholamine synthesis from [(14)C]tyrosine without any change in catecholamine secretion. These findings suggest that leptin stimulates catecholamine synthesis through its receptors in bovine adrenal medullary cells.

Adrenal Medulla↗

PubMed2000

Inhibitory effects of clozapine and other antipsychotic drugs on noradrenaline transporter in cultured bovine adrenal medullary cells.

The effects of clozapine and other antipsychotic drugs on noradrenaline (NA) transport were examined in cultured bovine adrenal medullary cells and in transfected Xenopus laevis oocytes expressing the bovine NA transporter. Incubation of adrenal medullary cells with clozapine (30-1000 ng/ml) inhibited desipramine (DMI)-sensitive uptake of [3H]NA in a concentration-dependent manner (IC50=110 ng/ml or 336 nM). Other antipsychotic drugs such as haloperidol, chlorpromazine, and risperidone also decreased [3H]NA uptake (IC50= 144, 220, and 210 ng/ml or 383, 690, and 512 nM, respectively). Eadie-Hofstee analysis showed that clozapine reduced V(max) of uptake of [3H]NA and increased K(m). Furthermore, clozapine inhibited specific binding of [3H]DMI to plasma membranes isolated from bovine adrenal medulla (IC50=48 ng/ml or 146 nM). Scatchard plot analysis of [3H]DMI binding revealed that clozapine decreased both B(max) and K(d). Other antipsychotic drugs, including haloperidol, chlorpromazine, and risperidone, also reduced [3H]DMI binding to the membranes. In transfected Xenopus oocytes expressing the bovine NA transporter, clozapine inhibited [3H]NA uptake in a concentration-dependent manner similar to that observed in adrenal medullary cells. These results suggest that clozapine and haloperidol directly inhibit transport of NA by acting on the site of an NA transporter that influences both substrate transport and binding of tricyclic antidepressants.

Adrenal Medulla↗

PubMed2000

Adrenomedullin inhibits spontaneous and bradykinin-induced but not oxytocin- or prostaglandin F(2alpha)-induced periodic contraction of rat uterus.

In isolated rat uterine strips, adrenomedullin (AM) inhibited the spontaneous periodic contraction in a concentration-dependent manner (IC(50)=22.3+/-0.7 nM). The inhibitory effect of AM was prevented by either AM(22-52), a putative antagonist for AM receptors, or calcitonin gene-related peptide (CGRP)(8-37), a putative antagonist for CGRP receptors. AM also attenuated bradykinin (BK)-induced periodic uterine contraction, which was blocked by AM(22-52) or CGRP(8-37), whereas AM had no effect on the periodic contraction caused by oxytocin or prostaglandin F(2alpha) (PGF(2alpha)). RT-PCR analysis showed that mRNAs for calcitonin receptor-like receptor (CRLR), receptor-activity-modifying protein (RAMP)1, RAMP2 and RAMP3 were expressed in the rat uterus. These results demonstrate that AM selectively inhibits spontaneous and BK-induced periodic contraction via activating receptors for AM and CGRP.

Adrenomedullin↗

PubMed2000

Heterologous facilitation of G protein-activated K(+) channels by beta-adrenergic stimulation via cAMP-dependent protein kinase.

To investigate possible effects of adrenergic stimulation on G protein-activated inwardly rectifying K(+) channels (GIRK), acetylcholine (ACh)-evoked K(+) current, I(KACh), was recorded from adult rat atrial cardiomyocytes using the whole cell patch clamp method and a fast perfusion system. The rise time of I(KACh ) was 0. 4 +/- 0.1 s. When isoproterenol (Iso) was applied simultaneously with ACh, an additional slow component (11.4 +/- 3.0 s) appeared, and the amplitude of the elicited I(KACh) was increased by 22.9 +/- 5.4%. Both the slow component of activation and the current increase caused by Iso were abolished by preincubation in 50 microM H89 (N-[2-((p -bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, a potent inhibitor of PKA). This heterologous facilitation of GIRK current by beta-adrenergic stimulation was further studied in Xenopus laevis oocytes coexpressing beta(2)-adrenergic receptors, m(2 )-receptors, and GIRK1/GIRK4 subunits. Both Iso and ACh elicited GIRK currents in these oocytes. Furthermore, Iso facilitated ACh currents in a way, similar to atrial cells. Cytosolic injection of 30-60 pmol cAMP, but not of Rp-cAMPS (a cAMP analogue that is inhibitory to PKA) mimicked the beta(2)-adrenergic effect. The possibility that the potentiation of GIRK currents was a result of the phosphorylation of the beta-adrenergic receptor (beta(2)AR) by PKA was excluded by using a mutant beta(2)AR in which the residues for PKA-mediated modulation were mutated. Overexpression of the alpha subunit of G proteins (Galpha(s)) led to an increase in basal as well as agonist-induced GIRK1/GIRK4 currents (inhibited by H89). At higher levels of expressed Galpha(s), GIRK currents were inhibited, presumably due to sequestration of the beta/gamma subunit dimer of G protein. GIRK1/GIRK5, GIRK1/GIRK2, and homomeric GIRK2 channels were also regulated by cAMP injections. Mutant GIRK1/GIRK4 channels in which the 40 COOH-terminal amino acids (which contain a strong PKA phosphorylation consensus site) were deleted were also modulated by cAMP injections. Hence, the structural determinant responsible is not located within this region. We conclude that, both in atrial myocytes and in Xenopus oocytes, beta-adrenergic stimulation potentiates the ACh-evoked GIRK channels via a pathway that involves PKA-catalyzed phosphorylation downstream from beta(2)AR.

Acetylcholine↗

PubMed2000

ATP activates DNA synthesis by acting on P2X receptors in human osteoblast-like MG-63 cells.

In human osteoblast-like MG-63 cells, extracellular ATP increased [(3)H]thymidine incorporation and cell proliferation and synergistically enhanced platelet-derived growth factor- or insulin-like growth factor I-induced [(3)H]thymidine incorporation. ATP-induced [(3)H]thymidine incorporation was mimicked by the nonhydrolyzable ATP analogs adenosine 5'-O-(3-thiotriphosphate) and adenosine 5'-adenylylimidodiphosphate and was inhibited by the P2 purinoceptor antagonist suramin, suggesting involvement of P2 purinoceptors. The P2Y receptor agonist UTP and UDP and a P2Y receptor antagonist reactive blue 2 did not affect [(3)H]thymidine incorporation, whereas the P2X receptor antagonist pyridoxal phosphate-6-azophenyl-2',4-disulfonic acid inhibited ATP-induced [(3)H]thymidine incorporation, suggesting that ATP-induced DNA synthesis was mediated by P2X receptors. RT-PCR analysis revealed that MG-63 cells expressed P2X(4), P2X(5), P2X(6), and P2X(7), but not P2X(1), P2X(2), and P2X(3), receptors. In fura 2-loaded cells, not only ATP, but also UTP, increased intracellular Ca(2+) concentration, and inhibitors for several Ca(2+)-activated protein kinases had no effect on ATP-induced DNA synthesis, suggesting that an increase in intracellular Ca(2+) concentration is not indispensable for ATP-induced DNA synthesis. ATP increased mitogen-activated protein kinase activity in a Ca(2+)-independent manner and synergistically enhanced platelet-derived growth factor- or insulin-like growth factor I-induced kinase activity. Furthermore, the mitogen-activated protein kinase kinase inhibitor PD-98059 totally abolished ATP-induced DNA synthesis. We conclude that ATP increases DNA synthesis and enhances the proliferative effects of growth factors through P2X receptors by activating a mitogen-activated protein kinase pathway.

Adenosine Triphosphate↗