Shimozono M, Liu J, Scofield M A, Wangemann P
Cell Physiology Laboratory, Boys Town National Research Hospital, Omaha, NE 68131, USA.
J Membr Biol. 1998 May 1;163(1):37-46. doi: 10.1007/s002329900368.
The transport of lactate and pyruvate across membranes of vestibular dark cells (VDC) may be important under aerobic, ischemic or hypoxic conditions. This study addresses the questions whether VDC from the gerbil contain an H+/monocarboxylate- cotransporter (MCT) and in which membrane, apical or basolateral, MCT is located. Uptake of monocarboxylates into VDC was monitored in functional studies by measuring the cytosolic pH (pHi) and by measuring the pH-sensitive equivalent short circuit current (Isc). Subtypes of the functionally identified MCT which are present in vestibular labyrinth tissues were identified as transcripts by cloning and sequencing of reverse-transcriptase polymerase chain reaction (RT-PCR) products. Monocarboxylates but not dicarboxylates induced a transient acidification of pHi which was inhibited by 5 mM alpha-cyano-4-hydroxycinnamate (CHC) but not by 1 microM DIDS or 500 microM pCMBS. The initial rate of acidification induced by monocarboxylates was dose-dependent in the range between 1 and 20 mM. K(m) values were for pyruvate 1.3, acetate 3.7, L-lactate 3.8 and D-lactate 7.3 mM. Both apical and basolateral application of monocarboxylates caused a transient increase of Isc which was sensitive to 5 mM CHC. RT-PCR revealed the presence of transcripts for the MCT subtypes MCT1 and MCT2. The identity of transcripts was confirmed by sequence analysis. These observations suggest that VDC contain an MCT in their apical and basolateral membrane and that the vestibular labyrinth contains transcripts for the subtypes MCT1 and MCT2.
在前庭暗细胞(VDC)膜上,乳酸和丙酮酸的转运在有氧、缺血或缺氧条件下可能具有重要意义。本研究探讨了沙鼠的VDC是否含有H⁺/单羧酸共转运体(MCT)以及MCT位于顶膜还是基底外侧膜这两个问题。在功能研究中,通过测量胞质pH(pHi)以及测量pH敏感的等效短路电流(Isc)来监测单羧酸进入VDC的摄取情况。通过逆转录聚合酶链反应(RT-PCR)产物的克隆和测序,将在前庭迷路组织中存在的功能鉴定的MCT亚型鉴定为转录本。单羧酸而非二羧酸会引起pHi的短暂酸化,这种酸化被5 mM α-氰基-4-羟基肉桂酸(CHC)抑制,但不被1 μM二异丁基氨磺酰苯(DIDS)或500 μM对氯汞苯甲酸(pCMBS)抑制。单羧酸诱导的酸化初始速率在1至20 mM范围内呈剂量依赖性。丙酮酸、乙酸、L-乳酸和D-乳酸的米氏常数(K(m))值分别为1.3 mM、3.7 mM、3.8 mM和7.3 mM。单羧酸的顶膜和基底外侧应用均导致Isc短暂增加,且该增加对5 mM CHC敏感。RT-PCR显示存在MCT亚型MCT1和MCT2的转录本。通过序列分析证实了转录本的身份。这些观察结果表明,VDC在其顶膜和基底外侧膜中含有MCT,并且前庭迷路含有MCT1和MCT2亚型的转录本。