功能性ghrelin受体在内脏迷走和脊髓传入神经通路中的表达
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摘要
目的：揭示功能性Ghrelin受体(GHS-R 1a)存在于支配大鼠胃肠道运动的迷走及脊髓内脏传入神经通路中以及探讨ghrelin是否参与这一通路的功能调节。
实验设计及方法：为了探讨功能性Ghrelin受体(GHS-RIa)在迷走结状神经节(NG nodose ganglion)及脊髓内脏传入神经通路背根神经节(DRG，dorsal rootganglia)中的存在及表达，我们采用原位杂交，免疫组织化学荧光染色及激光共聚焦显微镜成像等技术，从mRNA和蛋白表达等角度观察了GHS-R1a在NG，DRG神经元及卫星胶质细胞(SGC)中的存在。为了识别此受体在有神经纤维投射到胃的脊髓传入神经DRG中的分布，我们应用神经逆行追踪技术，首先将示踪物荧光金(Fluor-gold，FG)注射到大鼠手术暴露的胃壁，5天后将DRG取出，然后用三重，即GHS-R1a抗体，CGRP(降钙素基因相关肽)抗体及荧光金染色，来识别存在于内脏投射神经中的功能性Ghrelin受体。为了阐明Ghrelin及Ghrelin受体在胃肠与大脑之间信息传递中的具体功能，即ghrelin能否激活脊髓内脏传入通路中的感觉受体，我们使用激光共聚焦显微镜(CLSM)在室温下(20℃)记录了从新生大鼠(2-14天)中急性分离的DRG神经元细胞体及卫星胶质细胞(SGC)胞内的Ca~(2+)浓度。通过灌注包括ghrelin，ghrelin拟似剂GHRP-6以及ghrelin拮抗剂(D-Lys3)-GHRP-6等在内的不同的药剂研究了Ghrelin受体的激活情况。
结果：(1)应用免疫荧光双标(GHS-R 1a与CGRP)及原位杂交从蛋白及mRNA水平证实了GHS-R1a存在于大鼠迷走传入神经通路NG及脊髓内脏通路DRG的神经元与卫星胶质细胞中。
(2)应用免疫双标技术发现在背根神经节和结状神经节中都有一些GHS-R1a免疫反应阳性神经元同时CGRP染色呈显阳性，显示GHS-R1a和CGRP共存于同一神经原中的现象，表明内脏传入神经元存在于许多亚核群，同时也进一步提示大鼠胃中ghrelin的表达和内脏传入神经中降钙素基因相关肽(CGRP)的表达可能是关联的。
(3)应用荧光金(Fluorogold)标记的神经逆行追踪技术从胃追踪到背根神经节和结状神经节的神经元进行免疫组织化学染色，发现一些表达CGRP的GHS-R1a免疫反应阳性神经元也被荧光金染色。
(4)DRG胞内钙离子浓度([Ca2+]i)动态变化的LSCM测定：持续给予生长激素释放肽—6(GHRP-6；10(-6)M；)30-60秒，有55％的神经元(52／95；18个神经节)产生迅速(平均延滞期为11秒)而短暂的胞内Ca~(2+)浓度增加。在测定的浓度范围内，反应幅度不依赖于GHRP-6的浓度(10~(-13)-10~(-5)M)变化，而以平均相对荧光强度(RF)来表示，其值为1.16±0.15RF(mean±SD)，相当于神经元对KCl灌流反应幅度的43％。几乎所有的(91％)活卫星胶质细胞(SGC)都对GHRP-6有反应，反应延滞期为11秒，幅度的平均相对荧光强度为1.62±0.38RF，相当于对KCl灌流反应幅度的103±59％。对GHRP-6发生反应的细胞百分率作S型量效反应关系曲线(10~(-14)-10~(-5) M)该曲线显示DRG神经元和SGC的EC_(50)均为10~(-11)M。有趣的是从外周神经节中得到的这个EC50和以前报道的ghrelin引发下丘脑弓状核神经元锋电位及胞内Ca~(2+)浓度变化的EC50也是一致的。持续灌流gherlin(rat)引起大鼠DRG神经元及SGC反应的EC_(50)也为10`(-11)M，即与对GHRP-6的反应的EC_(50)一致。正如所预期的一样，ghrelin无功能的同源异构体(Des-octanoyl)-Gherkin(human，10~(-10)-10~(-10)M)并不能引起[Ca2+]i的增加。但是持续灌流Ghrelin受体拮抗剂(D-Lys3)-GHRP-6(10~(-6) to 10~(-12)M) 30秒却能够引起DRG神经元细胞体及SGC[Ca2+]i增加，因此在此实验中并没有观察到它对Ghrelin的拮抗作用。
结论：这些结果不但证实了GHS-R1a在大鼠迷走传入神经通路NG中的表达，而且首次表明脊髓传入神经通路DRG中的神经细胞及卫星胶质细胞有功能性ghrelin受体的存在，并进一步揭示相当于生理剂量的ghrelin(≥10~(-10)M)对这两种细胞有兴奋作用。这些发现支持了卫星胶质细胞的化学感受效应并且同时也预示了ghrelin对内脏脊髓信号的调制作用。
Objectives: The present study aims to unravel the presence of functional GHS-R 1a in the innervation pathways of the gastrointestinal tract of the rat, and to further elucidate the role of peripheral ghrelin in the vagal and spinal visceral afferent pathways involved in sensory reception.
Material and Methods: In this study we examined the expression of the functional GHS-R 1a in nodose ganglion (NG), dorsal root ganglion (DRG) nurons and satellite glial cells(SGC) of the rat in mRNA level (in situ hybridization) and at protein level (immunocytochemistry). The extrinsic primary afferent visceroceptive DRG and NG neurons were identified with retrograde tracing fluorogold from the stomach and stained for GHS-Rla and CGRP to determine the expression of GHS-R1a. To further investigate the role of ghrelin in the spinal visceral afferent pathway involved in sensory reception, we used a Confocal Laser Scanning Microscope (CLSM) (one image/s) to monitor the cytosolic Ca~(2+). concentration ([Ca2+]i)of neurons and satellite glial cells (SGC) in freshly isolated dorsal root ganglia (D2-D14; T8-T13) loaded with Fluo-4-AM (10~(-6) M; 40 min). [Ca2+]i was recorded respectively after application of ghrelin, ghrelin mimic GHRP-6 and ghrelin antagonist D-Lys-GHRP-6).
Results:
(1) The presence of the ghrelin receptor (GHS-R1a) in nodose ganglion and dorsal root ganglion neurons and SGC was demonstrated at the mRNA level (in situ hybridization). and at the protein level (immunocytochemistry, using double-labeling with CGRP to identify viscerally projecting neurons).
(2) GHS-R 1a was also co-localized with CGRP in some neurons in both DRG and NG, indicating the existence of subpopulations of visceral afferents.
(3)Some CGRP-expressing GHS-R 1a immunoreacitve neurons were found containing the retrograde labeled FG.
(4) Optical Recording of Cytosolic [Ca~(2+)]:
About 55% of the neurons (52/95; 18 ganglia) responded to GH-releasing peptide (GHRP-6; 10~(-6) M; application 30-60s) with a fast (mean lag time 11 s) and transient rise in [Ca~(2+)]. The amplitude of the responses was independent on the concentration of GHRP-6 (10~(-13) to 10~(-5) M) and was on average 1.16±0.15 RF (Relative Fluorescence; mean±SD), being 43% of the KCl response. Almost all (91%) SGC responded to GHRP-6, with a lag time of 11 s and amplitude equal to (103±59%; n=98) the response to KCl(1.62±0.38 RF). Sigmoid dose-response relations (10~(-14) to 10~(-5) M), based on the % of responding cells, revealed an EC_(50) of 10~(-11) M for both neurons and SGC. Interestingly, this EC_(50) for the peripherally located ganglion is comparable to previously reported effects of ghrelin on spike activity and [Ca~(2+)] of the hypothalamic arcuate nucleus neurons. Application of ghrelin (rat) did evoke responses in the neurons and SGC comparable to those to GHRP-6 (EC_(50) 10~(-11) M). (Des-octanoyl)-ghrelin (human; range 10~(-11) to 10~(-7) M) did not evoke responses.
Conclusion: Our results not only demonstrate the expression of GHS-R 1a in vagal afferents but also provide the first and direct morphological evidence of its presence in spinal visceral afferents. Our results further indicate that ghrelin, in concentrations comparable to those reported in plasma (= 10~(-10) M), has an excitatory effect on both ganglionic cell types. These findings support the notion of a chemosensory role of the SGC and indicate a modulating role of ghrelin in visceral spinal signaling.
实验设计及方法：为了探讨功能性Ghrelin受体(GHS-RIa)在迷走结状神经节(NG nodose ganglion)及脊髓内脏传入神经通路背根神经节(DRG，dorsal rootganglia)中的存在及表达，我们采用原位杂交，免疫组织化学荧光染色及激光共聚焦显微镜成像等技术，从mRNA和蛋白表达等角度观察了GHS-R1a在NG，DRG神经元及卫星胶质细胞(SGC)中的存在。为了识别此受体在有神经纤维投射到胃的脊髓传入神经DRG中的分布，我们应用神经逆行追踪技术，首先将示踪物荧光金(Fluor-gold，FG)注射到大鼠手术暴露的胃壁，5天后将DRG取出，然后用三重，即GHS-R1a抗体，CGRP(降钙素基因相关肽)抗体及荧光金染色，来识别存在于内脏投射神经中的功能性Ghrelin受体。为了阐明Ghrelin及Ghrelin受体在胃肠与大脑之间信息传递中的具体功能，即ghrelin能否激活脊髓内脏传入通路中的感觉受体，我们使用激光共聚焦显微镜(CLSM)在室温下(20℃)记录了从新生大鼠(2-14天)中急性分离的DRG神经元细胞体及卫星胶质细胞(SGC)胞内的Ca~(2+)浓度。通过灌注包括ghrelin，ghrelin拟似剂GHRP-6以及ghrelin拮抗剂(D-Lys3)-GHRP-6等在内的不同的药剂研究了Ghrelin受体的激活情况。
结果：(1)应用免疫荧光双标(GHS-R 1a与CGRP)及原位杂交从蛋白及mRNA水平证实了GHS-R1a存在于大鼠迷走传入神经通路NG及脊髓内脏通路DRG的神经元与卫星胶质细胞中。
(2)应用免疫双标技术发现在背根神经节和结状神经节中都有一些GHS-R1a免疫反应阳性神经元同时CGRP染色呈显阳性，显示GHS-R1a和CGRP共存于同一神经原中的现象，表明内脏传入神经元存在于许多亚核群，同时也进一步提示大鼠胃中ghrelin的表达和内脏传入神经中降钙素基因相关肽(CGRP)的表达可能是关联的。
(3)应用荧光金(Fluorogold)标记的神经逆行追踪技术从胃追踪到背根神经节和结状神经节的神经元进行免疫组织化学染色，发现一些表达CGRP的GHS-R1a免疫反应阳性神经元也被荧光金染色。
(4)DRG胞内钙离子浓度([Ca2+]i)动态变化的LSCM测定：持续给予生长激素释放肽—6(GHRP-6；10(-6)M；)30-60秒，有55％的神经元(52／95；18个神经节)产生迅速(平均延滞期为11秒)而短暂的胞内Ca~(2+)浓度增加。在测定的浓度范围内，反应幅度不依赖于GHRP-6的浓度(10~(-13)-10~(-5)M)变化，而以平均相对荧光强度(RF)来表示，其值为1.16±0.15RF(mean±SD)，相当于神经元对KCl灌流反应幅度的43％。几乎所有的(91％)活卫星胶质细胞(SGC)都对GHRP-6有反应，反应延滞期为11秒，幅度的平均相对荧光强度为1.62±0.38RF，相当于对KCl灌流反应幅度的103±59％。对GHRP-6发生反应的细胞百分率作S型量效反应关系曲线(10~(-14)-10~(-5) M)该曲线显示DRG神经元和SGC的EC_(50)均为10~(-11)M。有趣的是从外周神经节中得到的这个EC50和以前报道的ghrelin引发下丘脑弓状核神经元锋电位及胞内Ca~(2+)浓度变化的EC50也是一致的。持续灌流gherlin(rat)引起大鼠DRG神经元及SGC反应的EC_(50)也为10`(-11)M，即与对GHRP-6的反应的EC_(50)一致。正如所预期的一样，ghrelin无功能的同源异构体(Des-octanoyl)-Gherkin(human，10~(-10)-10~(-10)M)并不能引起[Ca2+]i的增加。但是持续灌流Ghrelin受体拮抗剂(D-Lys3)-GHRP-6(10~(-6) to 10~(-12)M) 30秒却能够引起DRG神经元细胞体及SGC[Ca2+]i增加，因此在此实验中并没有观察到它对Ghrelin的拮抗作用。
结论：这些结果不但证实了GHS-R1a在大鼠迷走传入神经通路NG中的表达，而且首次表明脊髓传入神经通路DRG中的神经细胞及卫星胶质细胞有功能性ghrelin受体的存在，并进一步揭示相当于生理剂量的ghrelin(≥10~(-10)M)对这两种细胞有兴奋作用。这些发现支持了卫星胶质细胞的化学感受效应并且同时也预示了ghrelin对内脏脊髓信号的调制作用。
Objectives: The present study aims to unravel the presence of functional GHS-R 1a in the innervation pathways of the gastrointestinal tract of the rat, and to further elucidate the role of peripheral ghrelin in the vagal and spinal visceral afferent pathways involved in sensory reception.
Material and Methods: In this study we examined the expression of the functional GHS-R 1a in nodose ganglion (NG), dorsal root ganglion (DRG) nurons and satellite glial cells(SGC) of the rat in mRNA level (in situ hybridization) and at protein level (immunocytochemistry). The extrinsic primary afferent visceroceptive DRG and NG neurons were identified with retrograde tracing fluorogold from the stomach and stained for GHS-Rla and CGRP to determine the expression of GHS-R1a. To further investigate the role of ghrelin in the spinal visceral afferent pathway involved in sensory reception, we used a Confocal Laser Scanning Microscope (CLSM) (one image/s) to monitor the cytosolic Ca~(2+). concentration ([Ca2+]i)of neurons and satellite glial cells (SGC) in freshly isolated dorsal root ganglia (D2-D14; T8-T13) loaded with Fluo-4-AM (10~(-6) M; 40 min). [Ca2+]i was recorded respectively after application of ghrelin, ghrelin mimic GHRP-6 and ghrelin antagonist D-Lys-GHRP-6).
Results:
(1) The presence of the ghrelin receptor (GHS-R1a) in nodose ganglion and dorsal root ganglion neurons and SGC was demonstrated at the mRNA level (in situ hybridization). and at the protein level (immunocytochemistry, using double-labeling with CGRP to identify viscerally projecting neurons).
(2) GHS-R 1a was also co-localized with CGRP in some neurons in both DRG and NG, indicating the existence of subpopulations of visceral afferents.
(3)Some CGRP-expressing GHS-R 1a immunoreacitve neurons were found containing the retrograde labeled FG.
(4) Optical Recording of Cytosolic [Ca~(2+)]:
About 55% of the neurons (52/95; 18 ganglia) responded to GH-releasing peptide (GHRP-6; 10~(-6) M; application 30-60s) with a fast (mean lag time 11 s) and transient rise in [Ca~(2+)]. The amplitude of the responses was independent on the concentration of GHRP-6 (10~(-13) to 10~(-5) M) and was on average 1.16±0.15 RF (Relative Fluorescence; mean±SD), being 43% of the KCl response. Almost all (91%) SGC responded to GHRP-6, with a lag time of 11 s and amplitude equal to (103±59%; n=98) the response to KCl(1.62±0.38 RF). Sigmoid dose-response relations (10~(-14) to 10~(-5) M), based on the % of responding cells, revealed an EC_(50) of 10~(-11) M for both neurons and SGC. Interestingly, this EC_(50) for the peripherally located ganglion is comparable to previously reported effects of ghrelin on spike activity and [Ca~(2+)] of the hypothalamic arcuate nucleus neurons. Application of ghrelin (rat) did evoke responses in the neurons and SGC comparable to those to GHRP-6 (EC_(50) 10~(-11) M). (Des-octanoyl)-ghrelin (human; range 10~(-11) to 10~(-7) M) did not evoke responses.
Conclusion: Our results not only demonstrate the expression of GHS-R 1a in vagal afferents but also provide the first and direct morphological evidence of its presence in spinal visceral afferents. Our results further indicate that ghrelin, in concentrations comparable to those reported in plasma (= 10~(-10) M), has an excitatory effect on both ganglionic cell types. These findings support the notion of a chemosensory role of the SGC and indicate a modulating role of ghrelin in visceral spinal signaling.
引文
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31. Bisschops R, Vanden Berghe P, Sarnelli G, Janssens J, Tack J. CRF-induced calcium signaling in guinea pig small intestine myenteric neurons involves CRF-1 receptors and activation of voltage-sensitive calcium channels. Am J Physiol Gastrointest Liver Physiol. 2006 Jun;290(6):G1252-60. Epub 2005 Dec 29.
32. Depoortere I, Thijs T, Peeters T. The contractile effect of the ghrelin receptor antagonist, D-Lys3-GHRP-6, in rat fundic strips is mediated through 5-HT receptors. Eur J Pharmacol. 2006 May 10;537(1-3): 160-5. Epub 2006 Mar 24.
1 Berridge MJ Neuronal calcium signaling. Neuron. 1998 Jul;21(1):13-26
2 Sparagna GC, Gunter KK, Sheu SS, Gunter TE. Mitochondrial calcium uptake from physiological-type pulses of calcium. A description of the rapid uptake mode. J Biol Chem. 1995 Nov 17;270(46):27510-5
3. Bernardi P. Mitochondria in muscle cell death. Ital J Neurol Sci. 1999 Dec;20(6):395-400.
4 Missiaen L, Dode L, Vanoevelen J, Raeymaekers L, Wuytack F. Calcium in the Golgi apparatus Cell Calcium. 2006 Nov 29; [Epub ahead of print]
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6. Vanoevelen J, Raeymaekers L, Dode L, Parys JB, De Smedt H, Callewaert G, Wuytack F, Missiaen L. Cytosolic Ca2+ signals depending on the functional state of the Golgi in HeLa cells. Cell Calcium. 2005 Nov;38(5):489-95
7. Takahashi N, Nemoto T, Kimura R, Tachikawa A, Miwa A, Okado H, Miyashita Y, lino M, Kadowaki T, Kasai H. Two-photon excitation imaging of pancreatic islets with various fluorescent probes. Diabetes. 2002 Feb;51 Suppl 1:S25-8.
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29. Traebert M, Riediger T, Whitebread S, Scharrer E, Schmid HA. Ghrelin acts on leptin-responsive neurones in the rat arcuate nucleus. J Neuroendocrinol. 2002 Jul;14(7):580-6
30. Pierno S, De Luca A, Desaphy JF, Fraysse B, Liantonio A, Didonna MP, Lograno M, Cocchi D, Smith RG, Camerino DC. Growth hormone secretagogues modulate the electrical and contractile properties of rat skeletal muscle through a ghrelin-specific receptor. Br J Pharmacol. 2003 Jun;139(3):575-84
31. Bisschops R, Vanden Berghe P, Sarnelli G, Janssens J, Tack J. CRF-induced calcium signaling in guinea pig small intestine myenteric neurons involves CRF-1 receptors and activation of voltage-sensitive calcium channels. Am J Physiol Gastrointest Liver Physiol. 2006 Jun;290(6):G1252-60. Epub 2005 Dec 29.
32. Depoortere I, Thijs T, Peeters T. The contractile effect of the ghrelin receptor antagonist, D-Lys3-GHRP-6, in rat fundic strips is mediated through 5-HT receptors. Eur J Pharmacol. 2006 May 10;537(1-3): 160-5. Epub 2006 Mar 24.
1 Berridge MJ Neuronal calcium signaling. Neuron. 1998 Jul;21(1):13-26
2 Sparagna GC, Gunter KK, Sheu SS, Gunter TE. Mitochondrial calcium uptake from physiological-type pulses of calcium. A description of the rapid uptake mode. J Biol Chem. 1995 Nov 17;270(46):27510-5
3. Bernardi P. Mitochondria in muscle cell death. Ital J Neurol Sci. 1999 Dec;20(6):395-400.
4 Missiaen L, Dode L, Vanoevelen J, Raeymaekers L, Wuytack F. Calcium in the Golgi apparatus Cell Calcium. 2006 Nov 29; [Epub ahead of print]
5. Van Baelen K, Dode L, Vanoevelen J, Callewaert G, De Smedt H, Missiaen L, Parys JB, Raeymaekers L, Wuytack F. The Ca2+/Mn2+ pumps in the Golgi apparatus. Biochim Biophys Acta. 2004 Dec 6;1742(1-3):103-12
6. Vanoevelen J, Raeymaekers L, Dode L, Parys JB, De Smedt H, Callewaert G, Wuytack F, Missiaen L. Cytosolic Ca2+ signals depending on the functional state of the Golgi in HeLa cells. Cell Calcium. 2005 Nov;38(5):489-95
7. Takahashi N, Nemoto T, Kimura R, Tachikawa A, Miwa A, Okado H, Miyashita Y, lino M, Kadowaki T, Kasai H. Two-photon excitation imaging of pancreatic islets with various fluorescent probes. Diabetes. 2002 Feb;51 Suppl 1:S25-8.
8. Shuttleworth TJ, Thompson JL, Mignen O. ARC channels: a novel pathway for receptor-activated calcium entry. Physiology (Bethesda). 2004 Dec;19:355-61
9. Vanden Berghe P, Kenyon JL, Smith TK. Mitochondrial Ca2+ uptake regulates the excitability of myenteric neurons. J Neurosci. 2002 Aug 15;22(16):6962-71
10. Vanden Berghe P, Molhoek S, Missiaen L, Tack J, Janssens J. Differential Ca(2+) signaling characteristics of inhibitory and excitatory myenteric motor neurons in culture. Am J Physiol Gastrointest Liver Physiol. 2000 Nov;279(5):G1121-7.
11. Hanani M, Wood JD. Corticotropin-releasing hormone excites myenteric neurons in the guinea-pig small intestine. Eur J Pharmacol. 1992 Jan 28;211(1):23-7.