Gu Y, Genever P G, Skerry T M, Publicover S J
School of Biosciences, University of Birmingham, P.O. Box 363, Birmingham B15 2TT, UK.
Calcif Tissue Int. 2002 Mar;70(3):194-203. doi: 10.1007/s00223-001-2004-z. Epub 2002 Feb 19.
Cells of mammalian bone express glutamate receptors. Functional N-methyl-D-aspartate (NMDA) receptors have been demonstrated in human, osteoblastic MG-63 cells, but currents in these cells, unlike those of mammalian neurons, are blocked by Mg(2+) in a voltage-insensitive manner. Differences between the characteristics of NMDA currents in bone cells and in neurons may reflect molecular variation of the receptors or associated molecules, with implications for the role(s) of glutamate in these different tissues and for targeting of ligands/antagonists. To determine whether NMDA receptors in primary bone cells are functional, and whether the currents carried by these receptors resemble those of MG-63 cells or those of mammalian neurons, we have applied the whole cell patch clamp technique to primary cultures of rat osteoblasts. In 0-Mg(2+) saline, 25% of cells showed a slowly developing inward current in response to bath perfusion with 1 mM or 100 microM NMDA. Antibodies against NMDA receptors stained approximately 26% of cells. When NMDA was applied by rapid superfusion, kinetics of the currents were similar to those of neuronal NMDA currents, reaching a peak within 20-30 ms. 1 mM Mg(2+) reduced current amplitude at negative holding potentials and caused the I-V relationship of the currents to adopt a 'J' shape rather than the linear relationship seen in the absence of added Mg(2+). Co-application of glycine (20 microM) with NMDA increased current amplitude by only 18%, suggesting that glycine is released from cells within the cultures. Currents were blocked by (+)-MK-801 and DL-2-amino-5-phosphonovaleric acid. Fluorimetric monitoring of Ca(2+) using fura-2 showed that, in Mg(2+)-free medium, NMDA caused a sustained rise in Ca(2+) that could be reversed by subsequent application of MK-801. We conclude that rat femoral osteoblasts express functional NMDA receptors and that these receptors differ from those previously identified in MG-63 cells. NMDA receptors of primary osteoblasts show a 'classical' voltage-sensitive Mg(2+) block, similar to that seen in neuronal NMDA receptors, and will therefore function as detectors of coincident receptor activation and membrane depolarization.
哺乳动物骨细胞表达谷氨酸受体。功能性N-甲基-D-天冬氨酸（NMDA）受体已在人成骨MG-63细胞中得到证实，但与哺乳动物神经元不同，这些细胞中的电流以电压不敏感的方式被Mg(2+)阻断。骨细胞和神经元中NMDA电流特性的差异可能反映了受体或相关分子的分子变异，这对谷氨酸在这些不同组织中的作用以及配体/拮抗剂的靶向性具有重要意义。为了确定原代骨细胞中的NMDA受体是否具有功能，以及这些受体所携带的电流是否类似于MG-63细胞或哺乳动物神经元的电流，我们将全细胞膜片钳技术应用于大鼠成骨细胞的原代培养。在无Mg(2+)的盐溶液中，25%的细胞在浴灌流1 mM或100 μM NMDA时显示出缓慢发展的内向电流。抗NMDA受体抗体对约26%的细胞进行了染色。当通过快速灌流施加NMDA时，电流动力学与神经元NMDA电流相似，在20 - 30毫秒内达到峰值。1 mM Mg(2+)在负钳制电位下降低了电流幅度，并使电流的I-V关系呈现出“J”形，而不是在未添加Mg(2+)时所见的线性关系。将甘氨酸（20 μM）与NMDA共同施加仅使电流幅度增加了18%，这表明甘氨酸是从培养物中的细胞释放出来的。电流被(+)-MK-801和DL-2-氨基-5-膦酸戊酸阻断。使用fura-2对Ca(2+)进行荧光监测表明，在无Mg(2+)的培养基中，NMDA导致Ca(2+)持续升高，随后施加MK-801可使其逆转。我们得出结论，大鼠股骨成骨细胞表达功能性NMDA受体，且这些受体与先前在MG-63细胞中鉴定的受体不同。原代成骨细胞的NMDA受体表现出“经典”的电压敏感性Mg(2+)阻断，类似于在神经元NMDA受体中所见，因此将作为同时发生的受体激活和膜去极化的检测器发挥作用。
