Linton S M, O'Donnell M J
Department of Biology, McMaster University, Hamilton, Ontario, Canada L8S 4K1.
J Exp Biol. 1999 Jun;202(Pt 11):1561-70. doi: 10.1242/jeb.202.11.1561.
Mechanisms of Na+ and K+ transport across the basolateral membrane of isolated Malpighian tubules of Drosophila melanogaster were studied by examining the effects of ion substitution and putative inhibitors of specific ion transporters on fluid secretion rates, basolateral membrane potential and secreted fluid cation composition. Inhibition of fluid secretion by [(dihydroindenyl)oxy]alkanoic acid (DIOA) and bumetanide (10(-)4 mol l-1) suggested that a K+:Cl- cotransporter is the main route for K+ entry into the principal cells of the tubules. Differences in the effects of bumetanide on fluxes of K+ and Na+ are inconsistent with effects upon a basolateral Na+:K+:2Cl- cotransporter. Large differences in electrical potential across apical (>100 mV, lumen positive) and basolateral (<60 mV, cell negative) cell membranes suggest that a favourable electrochemical gradient for Cl- entry into the cell may be used to drive K+ into the cell against its electrochemical gradient, via a DIOA-sensitive K+:Cl- cotransporter. A Na+/K+-ATPase was also present in the basolateral membrane of the Malpighian tubules. Addition of 10(-)5 to 10(-)3 mol l-1 ouabain to unstimulated tubules depolarized the basolateral potential, increased the Na+ concentration of the secreted fluid by 50-73 % and increased the fluid secretion rate by 10-19 %, consistent with an increased availability of intracellular Na+. We suggest that an apical vacuolar-type H+-ATPase and a basolateral Na+/K+-ATPase are both stimulated by cyclic AMP. In cyclic-AMP-stimulated tubules, K+ entry is stimulated by the increase in the apical membrane potential, which drives K+:Cl- cotransport at a faster rate, and by the stimulation of the Na+/K+-ATPase. Fluid secretion by cyclic-AMP-stimulated tubules was reduced by 26 % in the presence of ouabain, suggesting that the Na+/K+-ATPase plays a minor role in K+ entry into the tubule cells. Malpighian tubules secreted a Na+-rich (150 mmol l-1) fluid at high rates when bathed in K+-free amino-acid-replete saline (AARS). Secretion in K+-free AARS was inhibited by amiloride and bafilomycin A1, but not by bumetanide or hydrochlorothiazide, which inhibit Na+:Cl- cotransport. There was no evidence for a Na+ conductance in the basolateral membrane of unstimulated or cyclic-AMP-stimulated tubules. Possible mechanisms of Na+ entry into the tubule cells include cotransport with organic solutes such as amino acids and glucose.
通过研究离子替代以及特定离子转运体的假定抑制剂对液体分泌速率、基底外侧膜电位和分泌液阳离子组成的影响，对黑腹果蝇离体马氏管基底外侧膜上钠和钾的转运机制进行了研究。[(二氢茚基)氧基]链烷酸（DIOA）和布美他尼（10⁻⁴ mol/L）对液体分泌的抑制作用表明，钾氯共转运体是钾进入肾小管主细胞的主要途径。布美他尼对钾和钠通量影响的差异与对基底外侧钠钾氯共转运体的影响不一致。顶端（>100 mV，管腔为正）和基底外侧（<60 mV，细胞为负）细胞膜之间的电位存在很大差异，这表明氯离子进入细胞的有利电化学梯度可用于通过DIOA敏感的钾氯共转运体逆其电化学梯度驱动钾进入细胞。马氏管的基底外侧膜中也存在钠钾ATP酶。向未受刺激的肾小管中添加10⁻⁵至10⁻³ mol/L的哇巴因会使基底外侧电位去极化，使分泌液中的钠浓度增加50 - 73%，并使液体分泌速率增加10 - 19%，这与细胞内钠可用性增加一致。我们认为顶端液泡型质子ATP酶和基底外侧钠钾ATP酶均受环磷酸腺苷刺激。在环磷酸腺苷刺激的肾小管中，钾的进入受到顶端膜电位增加的刺激，这会以更快的速率驱动钾氯共转运，同时也受到钠钾ATP酶的刺激。在存在哇巴因的情况下，环磷酸腺苷刺激的肾小管的液体分泌减少了26%，这表明钠钾ATP酶在钾进入肾小管细胞过程中起次要作用。当浸泡在无钾且富含氨基酸的盐溶液（AARS）中时，马氏管会以高速率分泌富含钠（150 mmol/L）的液体。无钾AARS中的分泌受到氨氯吡咪和巴弗洛霉素A1的抑制，但不受抑制钠氯共转运的布美他尼或氢氯噻嗪的抑制。在未受刺激或环磷酸腺苷刺激的肾小管的基底外侧膜中没有钠电导的证据。钠进入肾小管细胞的可能机制包括与氨基酸和葡萄糖等有机溶质的共转运。
