Rat renal proximal tubular gluconeogenesis: possible involvement of nonmitochondrial carbonic anhydrase isozymes.
Rat renal proximal tubular gluconeogenesis: possible involvement of nonmitochondrial carbonic anhydrase isozymes.
复制标题
大鼠肾近端肾小管糖异生：非线粒体碳酸酐酶同工酶可能参与。
DOI：
10.1016/0003-9861(90)90079-e
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发表时间：
1990
影响因子：
3.9
通讯作者：
Cherian,K
中科院分区：
文献类型：
--
作者：
Dodgson,SJ;Cherian,K
碳酸酐酶（CA）抑制剂乙氧唑胺（ethoxzolamide）通过在25 mmHCO3 -中培养小管，而不是在50 mmHCO3 -中，降低葡萄糖从10 mmHCO3 -中合成的速率：这证明大鼠肾皮质线粒体CA （CA V）在生理总CO2（CO2+ HCO3 -）下为肾小管糖异生中丙酮酸羧化提供HCO3 -。在饥饿48小时的大鼠制备的肾近端小管中，用10毫米丙酮酸盐和25毫米hco3−缓冲盐水（Krebs-Henseleit缓冲液）孵育，CA抑制剂acetazolamide （AZ）和benzolamide （BZ）降低了葡萄糖合成的速度。125 μmAZ和450 μmBZ的抑制作用最大。丙酮酸浓度从3.33 mm增加到20 mm，葡萄糖合成速率随浓度的增加而增加；600 μmBZ或188 μmAZ时，葡萄糖合成不再依赖于丙酮酸浓度的增加。加倍碳酸氢盐的生理浓度恢复了葡萄糖合成对丙酮酸浓度的依赖，部分但不完全减轻了AZ和BZ的抑制作用，从而得出AZ和BZ除了影响CAV外，还通过影响酶来影响糖异生的结论。小管与不需要丙酮酸羧化的底物孵育以合成草酰乙酸。当小管在10毫米苹果酸盐中孵育时，葡萄糖合成速率不受低于100 μmAZ或400 μmBZ的影响，在125 μmAZ、450 μmBZ和更高浓度的药物浓度下，葡萄糖合成速率分别最大降低40%和20%。苹果酸浓度从3.33增加到20 min，葡萄糖合成速率降低；600 μmBZ仅在苹果酸浓度大于10 mm时抑制葡萄糖合成速率，而188 μmAZ在各浓度苹果酸浓度下均降低葡萄糖合成速率。当小管与CA抑制剂在谷氨酰胺中孵育时，结果相似。除600 μmBZ外，葡萄糖合成速率随底物代谢和底物浓度的不同而不同。由此可见，只有当丙酮酸被羧化时，BZ对cav的抑制作用才会影响线粒体草酰乙酸的产生。进一步得出结论，用BZ观察到的葡萄糖合成的最大减少是由于磷酸烯醇丙酮酸羧激酶（PEPCK）后糖异生的阻断。当浓度为188 μmAZ时，对葡萄糖合成速率有较大的抑制作用。这种抑制与底物浓度无关，表明后路阻滞；然而，葡萄糖的合成速率随每种底物而变化，表明在PEPCK之前有一个额外的阻滞。综上所述，AZ和BZ可抑制除CA v外参与糖异生的酶。由于大鼠肾小管中95%的CA活性是非线粒体的，因此表明这种抑制可能是抑制其他CA同工酶的继发效应。
The carbonic anhydrase (CA) inhibitor ethoxzolamide decreases the rate of glucose synthesis from 10 mmpyruvate by tubules incubating in 25 mmHCO3−but not in 50 mmHCO3−: this is evidence that rat renal cortical mitochondrial CA (CA V) provides HCO3−for pyruvate carboxylation in renal tubular gluconeogenesis at physiological total CO2(CO2+ HCO3−). In renal proximal tubules prepared from 48-h-starved rats and incubating in 10 mmpyruvate in 25 mmHCO3−buffered saline (Krebs-Henseleit buffer) the CA inhibitors acetazolamide (AZ) and benzolamide (BZ) decreased the rate of glucose synthesis. Maximal inhibition was reached with 125 μmAZ or with 450 μmBZ. The rate of glucose synthesis increased with increasing pyruvate concentration from 3.33 to 20 mm; including 600 μmBZ or 188 μmAZ results in glucose synthesis becoming independent of increasing pyruvate concentration. Doubling the physiological concentration of bicarbonate restored the dependence of glucose synthesis on pyruvate concentration and partly, but not completely, alleviated the inhibitory effect of AZ and BZ, leading to the conclusion that AZ and BZ influence gluconeogenesis by affecting enzymes in addition to CAV. Tubules were incubated with substrates which do not require pyruvate carboxylation for synthesis of oxaloacetate. When tubules were incubated in 10 mmmalate the rate of glucose synthesis was unaffected by less than 100 μmAZ or 400 μmBZ and was decreased maximally by 40 and 20%, respectively, by 125 μmAZ, 450 μmBZ, and higher concentrations of these drugs. Increasing the malate concentration from 3.33 to 20 mmincreased the rate of glucose synthesis; 600 μmBZ inhibited the rate of glucose synthesis only when the malate concentration was greater than 10 mmbut 188 μmAZ decreased the rate of glucose synthesis at each concentration of malate. Results were similar when tubules were incubated in glutamine with CA inhibitors. The rate of glucose synthesis differed with the substrate metabolized and the substrate concentration except when 600 μmBZ was included. It is concluded that only when pyruvate is carboxylated does BZ inhibition of CA V affect mitochondrial production of oxaloacetate. It is further concluded that the maximum decrease of glucose synthesis observed with BZ results from a block in gluconeogenesis after phosphoenolpyruvate carboxykinase (PEPCK). Greater inhibition of the rate of glucose synthesis was always observed with 188 μmAZ. This inhibition was independent of substrate concentration, indicating a posterior block; however, the rate of glucose synthesis varied with each substrate, indicating an additional block before PEPCK. It is concluded that AZ and BZ inhibit enzymes involved in gluconeogenesis other than CA V. Since 95% of the CA activity of rat kidney tubules is nonmitochondrial it is suggested that this inhibition may be a secondary effect of inhibition of other CA isozymes.