Kim Y C, Kim S J, Sim J H, Jun J Y, Kang T M, Suh S H, So I, Kim K W
Department of Physiology and Biophysics, Seoul National University College of Medicine, 28 Yongon-Dong, Chongno-Gu, Seoul 110-799, Korea.
Pflugers Arch. 1998 Jun;436(1):1-8. doi: 10.1007/s004240050597.
The possibility of the protein kinase C (PKC) pathway being a mechanism underlying the desensitization of carbachol- (CCh-)activated nonselective cationic current (ICCh) was investigated in a study of guinea-pig gastric myocytes. Using the conventional whole-cell patch-clamp technique with symmetrical CsCl-rich solution in pipette and bath, ICCh was induced by bath application of 50 microM CCh. With 0.5 mM EGTA [ethyleneglycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] in the pipette solution (0.5 mM [EGTA]i), ICCh decayed spontaneously (desensitization of ICCh) to around 20% within 10 min. Desensitization of ICCh was significantly attenuated with 2 mM [EGTA]i. At a concentration of 20 microM OAG (1-oleoyl-2-acetyl-sn-glycerol), a PKC activator, inhibited ICCh at 0.5 mM [EGTA]i but far less at 2 mM [EGTA]i (18% and 81% of control, respectively). The same cationic current induced by intracellular guanosine-5'-O-(3-thiotriphosphate) (GTP[gamma-S]) was not inhibited by OAG with 0.5 mM [EGTA]i. The pretreatment of gastric myocytes with PKC inhibitors, either 1 microM chelerythrine or 1 microM peptide inhibitor, attenuated the desensitization of ICCh. [Ca2+]i was also measured by single cell microfluorometry using fura-2. Under CCh stimulation with 2 mM [EGTA]i, [Ca2+]i did not increase above 100 nM while it increased to around 260 nM with 0.5 mM [EGTA]i. These results suggest that the desensitization of ICCh is partly due to the Ca2+-dependent PKC pathway in guinea-pig gastric myocytes.
在一项对豚鼠胃肌细胞的研究中，研究了蛋白激酶C（PKC）途径作为卡巴胆碱（CCh）激活的非选择性阳离子电流（ICCh）脱敏潜在机制的可能性。使用传统的全细胞膜片钳技术，移液管和浴槽中均使用富含CsCl的对称溶液，通过在浴槽中施加50微摩尔/升的CCh来诱导ICCh。移液管溶液中含有0.5毫摩尔/升的EGTA（乙二醇双（β-氨基乙基醚）-N，N，N'，N'-四乙酸）（0.5毫摩尔/升[EGTA]i）时，ICCh会在10分钟内自发衰减（ICCh脱敏）至约20%。2毫摩尔/升[EGTA]i可显著减弱ICCh的脱敏。在浓度为20微摩尔/升的OAG（1-油酰基-2-乙酰基-sn-甘油）（一种PKC激活剂）作用下，0.5毫摩尔/升[EGTA]i时可抑制ICCh，但2毫摩尔/升[EGTA]i时抑制作用小得多（分别为对照的18%和81%）。细胞内鸟苷-5'-O-（3-硫代三磷酸）（GTP[γ-S]）诱导的相同阳离子电流在0.5毫摩尔/升[EGTA]i时不受OAG抑制。用PKC抑制剂（1微摩尔/升白屈菜红碱或1微摩尔/升肽抑制剂）预处理胃肌细胞，可减弱ICCh的脱敏。还使用fura-2通过单细胞显微荧光测定法测量[Ca2+]i。在2毫摩尔/升[EGTA]i的CCh刺激下，[Ca2+]i不会升高至100纳摩尔以上，而在0.5毫摩尔/升[EGTA]i时会升高至约260纳摩尔。这些结果表明，豚鼠胃肌细胞中ICCh的脱敏部分归因于Ca2+依赖的PKC途径。
