三磷酸腺苷在新生大鼠耳蜗血管纹缘细胞中的释放机制
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摘要
目的探究新生大鼠耳蜗血管纹缘细胞中ATP释放的机制。方法原代培养新生SD大鼠血管纹缘细胞,利用钙离子荧光探针、生物发光法测定ATP浓度和β-氨基己糖苷酶释放率测定法来研究ATP和GPN与P2YR-PLC-IP3通路诱导的内质网钙库反应之间的关系,以及内质网钙库与细胞外ATP浓度和β-氨基己糖苷酶释放率的关系。结果 ATP和GPN可通过P2YR-PLC-IP3通路诱发内质网钙库释放,可被P2YR-PLC-IP3通路拮抗剂抑制。缘细胞外ATP浓度和β-氨基己糖苷酶释放率与细胞内钙离子浓度呈正相关。结论缘细胞外ATP作用于P2Y嘌呤能信号系统触发内质网钙库释放,胞内钙离子诱导溶酶体胞吐作用释放ATP至胞外,从而发挥其生物学作用。
Objective To elucidate mechanisms of ATP release from cochlear stria vascularis marginal cells in neonatal rats. Methods Stria vascularis marginal cells from 1 day old Sprague Dawley rats were cultured. Fluo-4 AM was used to investigate the influence of ATP and GPN on P2YR-PLC-IP3 pathway induced endoplasmic reticulum(ER) Ca~(2+) responses. ATP bioluminescence assay and β-hexosaminidase secretion assay were used to investigate the relationship between ER Ca~(2+) responses and extracellular ATP and β-hexosaminidase. Results ATP and GPN induced Ca~(2+) release from ER via the P2YR-PLC-IP3 pathway, which was inhibited by P2Y receptor antagonists. Release of ATP and β-hexosaminidase from marginal cells were dependent on ER Ca~(2+) responses. Conclusion Extracellular ATP can evoke ER Ca~(2+) responses via the P2Y-PLC-IP3 pathway. ER Ca~(2+) contributes to ATP release from lysosomes via exocytosis which helps to control Ca~(2+) homeostasis in marginal cells.
Objective To elucidate mechanisms of ATP release from cochlear stria vascularis marginal cells in neonatal rats. Methods Stria vascularis marginal cells from 1 day old Sprague Dawley rats were cultured. Fluo-4 AM was used to investigate the influence of ATP and GPN on P2YR-PLC-IP3 pathway induced endoplasmic reticulum(ER) Ca~(2+) responses. ATP bioluminescence assay and β-hexosaminidase secretion assay were used to investigate the relationship between ER Ca~(2+) responses and extracellular ATP and β-hexosaminidase. Results ATP and GPN induced Ca~(2+) release from ER via the P2YR-PLC-IP3 pathway, which was inhibited by P2Y receptor antagonists. Release of ATP and β-hexosaminidase from marginal cells were dependent on ER Ca~(2+) responses. Conclusion Extracellular ATP can evoke ER Ca~(2+) responses via the P2Y-PLC-IP3 pathway. ER Ca~(2+) contributes to ATP release from lysosomes via exocytosis which helps to control Ca~(2+) homeostasis in marginal cells.
引文
1 Mammano F.ATP-dependent intercellular Ca2+signaling in the developing cochlea:facts,fantasies and perspectives[J].Semin Cell Dev Biol,2013,24(1):31-39.
2 Piazza V,Ciubotaru C D,Galr J E,et al.Purinergic signalling and intercellular Ca 2+wave propagation in the organ of Corti[J].Cell Calcium,2007,41(1):77-86.
3 Mammano F..ATP-dependent intercellular Ca 2+signaling in the developing cochlea:Facts,fantasies and perspectives[J].Seminars in Cell&Developmental Biology,2013,24(1):31-39.
4何圆圆,杨军.新生大鼠耳蜗K(o)lliker器支持细胞ATP释放的机制[J].中华耳鼻咽喉头颈外科杂志,2015,50(1):43-49.He YY,Yang J.ATP release mechanism from the supporting cells in the K(o)lliker organ in vitro in the cochlea of newborn rat[J]Chinese Journal of Otorhinolaryngology Head and Neck Surgery,2015,50(1):43-49.
5陈洁,何珊,杨军.新生大鼠耳蜗血管纹缘细胞中ATP存在的证据[J].临床耳鼻咽喉头颈外科杂志,2010,24(10):462-465.Chen J,He S,Yang J.Evidence for the existence of ATP in the marginal cells of the neonatal rat cochlea[J]JOURNAL OFCLINICAL OTORHINOLARYNGOLOGY HEAD AND NECKSURGERY,2010,24(10):462-465.
6 Liu J,Liu W,Yang J.ATP-containing vesicles in stria vascular marginal cell cytoplasms in neonatal rat cochlea are lysosomes[J].Sci Rep,2016,6:20903.
7常青,龚树生.P2嘌呤受体在内耳中的分布特性和功能[J].国际耳鼻咽喉头颈外科杂志,2005,29(4):204-207.Chang Q,Gong SH.Distribution and function of P2 purine receptor in inner ear[J].International Journal of Otolaryngology-Head and Neck Surgery,2005,29(4):204-207.
8 Liu W J,Yang J.Developmental Expression of Inositol 1,4,5-trisphosphate Receptor in the Post-Natal Rat Cochlea[J].European Journal of Histochemistry,2015,59(2).
9 Kotova P D,Bystrova M F,Rogachevskaja O A,et al.Coupling of P2Y receptors to Ca(2+)mobilization in mesenchymal stromal cells from the human adipose tissue[J].Cell Calcium,2018,71:1-14.
10 Rutter G A,Hodson D J,Chabosseau P,et al.Local and regional control of calcium dynamics in the pancreatic islet[J].Diabetes Obes Metab,2017,19 Suppl 1:30-41.
11 Dou Y,Wu H-J,Li H-Q,et al.Microglial migration mediated by ATP-induced ATP release from lysosomes[J].Cell Research,2012,22:1022.
12 Sivaramakrishnan V,Bidula S,Campwala H,et al.Constitutive lysosome exocytosis releases ATP and engages P2Y receptors in human monocytes[J].J Cell Sci,2012,125(Pt 19):4567-4575.
13 Chen J,Zhu Y,Liang C,et al.Pannexin1 channels dominate ATPrelease in the cochlea ensuring endocochlear potential and auditory receptor potential generation and hearing[J].Sci Rep,2015,5:10762.
14塞娜,张桐,吴军,等.小鼠耳蜗血管纹血-迷路屏障免疫组织化学研究[J].中华耳科学杂志(英文版),2018,16(2):205-.Sai N,Zhang T,Wu J,et al.An Immunohistochemical Study of Cochlear Blood-labyrinth Barrier in Mice.Chinese Journal of Otology,2018,16(2):205-.
15 White P N,Thorne P R,Housley G D,et al.Quinacrine staining of marginal cells in the stria vascularis of the guinea-pig cochlea:a possible source of extracellular ATP?[J].Hearing Research,1995,90(1-2):97-105.
16 Zhang Z,Chen G,Zhou W,et al.Regulated ATP release from astrocytes through lysosome exocytosis[J].Nature Cell Biology,2007,9(8):945-953.
17 Gossman D G,Zhao H B.Hemichannel-mediated inositol 1,4,5-trisphosphate(IP3)release in the cochlea:a novel mechanism of IP3 intercellular signaling[J].Cell Commun Adhes,2008,15(4):305-315.
18 Huang L C,Thorne P R,Vlajkvic S M,et al.Differential expression of P2Y receptors in the rat cochlea during development[J].Purinergic Signal,2010,6(2):231-248.
19 Sage C L,Marcus D C.Immunolocalization of P2Y4 and P2Y2purinergic receptors in strial marginal cells and vestibular dark cells[J].J Membr Biol,2002,185(2):103-115.
20 Liu W J,Yang J.Developmental expression of inositol 1,4,5-trisphosphate receptor in the post-natal rat cochlea[J].Eur J Histochem,2015,59(2):2486.
21 Von Kugelgen I.Pharmacological profiles of cloned mammalian P2Y-receptor subtypes[J].Pharmacol Ther,2006,110(3):415-432.
22 Hatano N,Ohya S,Imaizumi Y,et al.ATP increases[Ca2+]i and activates a Ca2+‐dependent Cl-current in rat ventricular fibroblasts[J].Experimental Physiology,2018,:.
23 Macmillan D,Kennnedy C,Mccarron J G.ATP inhibits Ins(1,4,5)P3-evoked Ca2+release in smooth muscle via P2Y1receptors[J].Journal of Cell Science,2012,125(21):5151-5158.
24 Patel S,Docampo R.Acidic calcium stores open for business:expanding the potential for intracellular Ca2+signaling[J].Trends in Cell Biology,2010,20(5):277-286.
25 Kilpatrick I B S,Eden E R,Schapira A H,et al.Direct mobilisation of lysosomal Ca2+triggers complex Ca2+signals[J].J Cell Sci,2013,126(Pt 1):60-66.
26 Liang Y,Huang L,Yang J.Differential expression of ryanodine receptor in the developing rat cochlea[J].European Journal of Histochemistry Ejh,2009,53(4):249.
27 Morton-Jones R T,Cannell M B,Jeyakumar L H,et al.Differential expression of ryanodine receptors in the rat cochlea[J].Neuroscience,2006,137(1):275-286.
28 Penny C J,Kilpatrick B S,Eden E R,et al.Coupling acidic organelles with the ER through Ca2microdomains at membrane contact sites[J].Cell Calcium,2015,58(4):387.
29 Harikumar P,Reeves J P.The lysosomal proton pump is electrogenic[J].Journal of Biological Chemistry,1983,258(17):10403.
30 Muoz D J,Kendrick I S,Rassam M,.,et al.Vesicular storage of adenosine triphosphate in the guinea-pig cochlear lateral wall and concentrations of ATP in the endolymph during sound exposure and hypoxia[J].Acta Oto-Laryngologica,2001,121(1):10-15.
31 Claudio Marcleo F,Milton Osmar A,Maria Isabel C.ATP is released from autophagic vesicles to the extracellular space in a VAMP7-dependent manner[J].Autophagy,2012,8(12):1741-1756.
32 Zhang B,Hou R,Zou Z,et al.Mechanically induced autophagy is associated with ATP metabolism and cellular viability in osteocytesin vitro[J].Redox Biology,2018,14:492-498.
2 Piazza V,Ciubotaru C D,Galr J E,et al.Purinergic signalling and intercellular Ca 2+wave propagation in the organ of Corti[J].Cell Calcium,2007,41(1):77-86.
3 Mammano F..ATP-dependent intercellular Ca 2+signaling in the developing cochlea:Facts,fantasies and perspectives[J].Seminars in Cell&Developmental Biology,2013,24(1):31-39.
4何圆圆,杨军.新生大鼠耳蜗K(o)lliker器支持细胞ATP释放的机制[J].中华耳鼻咽喉头颈外科杂志,2015,50(1):43-49.He YY,Yang J.ATP release mechanism from the supporting cells in the K(o)lliker organ in vitro in the cochlea of newborn rat[J]Chinese Journal of Otorhinolaryngology Head and Neck Surgery,2015,50(1):43-49.
5陈洁,何珊,杨军.新生大鼠耳蜗血管纹缘细胞中ATP存在的证据[J].临床耳鼻咽喉头颈外科杂志,2010,24(10):462-465.Chen J,He S,Yang J.Evidence for the existence of ATP in the marginal cells of the neonatal rat cochlea[J]JOURNAL OFCLINICAL OTORHINOLARYNGOLOGY HEAD AND NECKSURGERY,2010,24(10):462-465.
6 Liu J,Liu W,Yang J.ATP-containing vesicles in stria vascular marginal cell cytoplasms in neonatal rat cochlea are lysosomes[J].Sci Rep,2016,6:20903.
7常青,龚树生.P2嘌呤受体在内耳中的分布特性和功能[J].国际耳鼻咽喉头颈外科杂志,2005,29(4):204-207.Chang Q,Gong SH.Distribution and function of P2 purine receptor in inner ear[J].International Journal of Otolaryngology-Head and Neck Surgery,2005,29(4):204-207.
8 Liu W J,Yang J.Developmental Expression of Inositol 1,4,5-trisphosphate Receptor in the Post-Natal Rat Cochlea[J].European Journal of Histochemistry,2015,59(2).
9 Kotova P D,Bystrova M F,Rogachevskaja O A,et al.Coupling of P2Y receptors to Ca(2+)mobilization in mesenchymal stromal cells from the human adipose tissue[J].Cell Calcium,2018,71:1-14.
10 Rutter G A,Hodson D J,Chabosseau P,et al.Local and regional control of calcium dynamics in the pancreatic islet[J].Diabetes Obes Metab,2017,19 Suppl 1:30-41.
11 Dou Y,Wu H-J,Li H-Q,et al.Microglial migration mediated by ATP-induced ATP release from lysosomes[J].Cell Research,2012,22:1022.
12 Sivaramakrishnan V,Bidula S,Campwala H,et al.Constitutive lysosome exocytosis releases ATP and engages P2Y receptors in human monocytes[J].J Cell Sci,2012,125(Pt 19):4567-4575.
13 Chen J,Zhu Y,Liang C,et al.Pannexin1 channels dominate ATPrelease in the cochlea ensuring endocochlear potential and auditory receptor potential generation and hearing[J].Sci Rep,2015,5:10762.
14塞娜,张桐,吴军,等.小鼠耳蜗血管纹血-迷路屏障免疫组织化学研究[J].中华耳科学杂志(英文版),2018,16(2):205-.Sai N,Zhang T,Wu J,et al.An Immunohistochemical Study of Cochlear Blood-labyrinth Barrier in Mice.Chinese Journal of Otology,2018,16(2):205-.
15 White P N,Thorne P R,Housley G D,et al.Quinacrine staining of marginal cells in the stria vascularis of the guinea-pig cochlea:a possible source of extracellular ATP?[J].Hearing Research,1995,90(1-2):97-105.
16 Zhang Z,Chen G,Zhou W,et al.Regulated ATP release from astrocytes through lysosome exocytosis[J].Nature Cell Biology,2007,9(8):945-953.
17 Gossman D G,Zhao H B.Hemichannel-mediated inositol 1,4,5-trisphosphate(IP3)release in the cochlea:a novel mechanism of IP3 intercellular signaling[J].Cell Commun Adhes,2008,15(4):305-315.
18 Huang L C,Thorne P R,Vlajkvic S M,et al.Differential expression of P2Y receptors in the rat cochlea during development[J].Purinergic Signal,2010,6(2):231-248.
19 Sage C L,Marcus D C.Immunolocalization of P2Y4 and P2Y2purinergic receptors in strial marginal cells and vestibular dark cells[J].J Membr Biol,2002,185(2):103-115.
20 Liu W J,Yang J.Developmental expression of inositol 1,4,5-trisphosphate receptor in the post-natal rat cochlea[J].Eur J Histochem,2015,59(2):2486.
21 Von Kugelgen I.Pharmacological profiles of cloned mammalian P2Y-receptor subtypes[J].Pharmacol Ther,2006,110(3):415-432.
22 Hatano N,Ohya S,Imaizumi Y,et al.ATP increases[Ca2+]i and activates a Ca2+‐dependent Cl-current in rat ventricular fibroblasts[J].Experimental Physiology,2018,:.
23 Macmillan D,Kennnedy C,Mccarron J G.ATP inhibits Ins(1,4,5)P3-evoked Ca2+release in smooth muscle via P2Y1receptors[J].Journal of Cell Science,2012,125(21):5151-5158.
24 Patel S,Docampo R.Acidic calcium stores open for business:expanding the potential for intracellular Ca2+signaling[J].Trends in Cell Biology,2010,20(5):277-286.
25 Kilpatrick I B S,Eden E R,Schapira A H,et al.Direct mobilisation of lysosomal Ca2+triggers complex Ca2+signals[J].J Cell Sci,2013,126(Pt 1):60-66.
26 Liang Y,Huang L,Yang J.Differential expression of ryanodine receptor in the developing rat cochlea[J].European Journal of Histochemistry Ejh,2009,53(4):249.
27 Morton-Jones R T,Cannell M B,Jeyakumar L H,et al.Differential expression of ryanodine receptors in the rat cochlea[J].Neuroscience,2006,137(1):275-286.
28 Penny C J,Kilpatrick B S,Eden E R,et al.Coupling acidic organelles with the ER through Ca2microdomains at membrane contact sites[J].Cell Calcium,2015,58(4):387.
29 Harikumar P,Reeves J P.The lysosomal proton pump is electrogenic[J].Journal of Biological Chemistry,1983,258(17):10403.
30 Muoz D J,Kendrick I S,Rassam M,.,et al.Vesicular storage of adenosine triphosphate in the guinea-pig cochlear lateral wall and concentrations of ATP in the endolymph during sound exposure and hypoxia[J].Acta Oto-Laryngologica,2001,121(1):10-15.
31 Claudio Marcleo F,Milton Osmar A,Maria Isabel C.ATP is released from autophagic vesicles to the extracellular space in a VAMP7-dependent manner[J].Autophagy,2012,8(12):1741-1756.
32 Zhang B,Hou R,Zou Z,et al.Mechanically induced autophagy is associated with ATP metabolism and cellular viability in osteocytesin vitro[J].Redox Biology,2018,14:492-498.