耗竭心肌细胞内糖原对缺血后再灌注心肌的保护作用及其机制

本工作在离体大鼠等容收缩心脏模型上观察耗竭心脏细胞内糖原对心肌缺血再灌注损伤的影响。心脏富氧灌流30 min后,随机分为三组:Ⅰ、富氧组:富氧灌流75min。Ⅱ、对照组:常规K-H液富氧灌流15min,旷置30min,再灌流30min。Ⅲ、耗竭糖原组:先用充N_2(95%N_2:5%CO_2)的K-H液灌流15min,余同组Ⅱ。结果表明。耗竭心肌细胞内糖原可提高再灌注后心脏血液动力学的恢复;冠脉流出液中LDH活性及心肌组织MDA含量降低,线粒体及胞浆液中GSH-Px有较高的活性;心肌组织Na~+,Ca~(2+)超负荷减轻。说明耗竭心肌细胞内糖原可通过减少细胞内H~+的生成抑制Na~+/H~+交换,从而明显减轻心肌缺血再灌注损伤。     

Amiloride对低血流缺氧再灌注心肌的保护作用

本文采用离体大鼠等容收缩心脏模型,探讨Na/H交换机制在心脏低血流缺氧后再灌注损伤中的意义。心脏富氧灌流30min后,随机分为三组:(1)富氧组:富氧灌流75 min;(2)对照组:低血流缺氧45 min后再恢复富氧灌流30 min;(3)Amiloride组:低血流缺氧期间,溶0.5(mmol/L)Amiloride(Na/H交换抑制剂)于K-H液中,余同组(2)。实验发现Amiloride组心脏各血液动力学指标优于对照组,冠脉流出液中LDH活性及心肌组织MDA含量均少于组(2),线粒体及胞浆液中GSH-Px活力得以保护,心肌组织Na~ 、Ca~(2 )超负荷减轻,K~ 丢失减少。结果表明:抑制Na~ /H~ 交换能明显减轻低血流缺氧心肌再灌注损伤。     

地高辛抗血清拮抗内洋地黄素介导的离体大鼠心肌缺氧复氧损伤的实验研究

目的：研究内洋地黄素在离体大鼠心脏缺氧复氧损伤中的变化，观察内洋地黄素特异性拮抗剂地高辛抗血清对大鼠心脏缺氧复氧损伤（A-RI）的拮抗作用。 方法： 制备离体大鼠心脏A-RI模型，60只SD 大鼠随机分为6组，每组10只。正常对照组：给予富氧K-H液灌流，流量10 mL·min-1，持续灌流90 min；A-RI组：富氧K-H液灌流30 min后，予以乏氧K-H液低流量（1-2 mL·min-1）灌流30 min，再给予富氧K-H液复灌30 min；维拉帕米组、小剂量、中剂量、大剂量地高辛抗血清组：于复氧前分别向灌流液中加入维拉帕米5 μg·kg-1、地高辛抗血清3.3 mg·kg-1、10 mg·kg-1、30 mg·kg-1，复氧灌流前灌注完，其余同A-RI组。各组于复氧灌流结束时，制备心肌匀浆，测定心肌匀浆中内洋地黄素含量、细胞膜Na+-K+-ATP酶活性以及线粒体总Ca2+水平，并观察心肌超微结构的变化。 结果： A-RI组心肌组织内洋地黄素水平明显高于正常对照组［（1.04±0.42）ng·g-1与（0.63±0.09）ng·g-1，P<0.01］，细胞膜Na+-K+-ATP酶活性明显低于正常对照组［（2.85±1.00) mmol·g-1Pr·h-1与（4.24±1.19)mmol·g-1Pr·h-1，P<0.01］，线粒体总Ca2+水平显著高于正常对照组［（0.368±0.113) μmol·g-1Pr·h-1与（0.130±0.004) μmol·g-1Pr·h-1，P<0.01］，心肌组织结构发生明显破坏。中、大剂量地高辛抗血清组心肌组织内洋地黄素水平显著低于A-RI组［（0.55±0.24)ng·g-1，(0.68±0.26) ng·g-1］，心肌组织Na+-K+-ATP酶活性显著高于A-RI组［（4.88±1.51)mmol·g-1Pr·h-1，（3.85±1.15)mmol·g-1Pr·h-1），心肌线粒体内总Ca2+含量显著低于A-RI组［（0.127±0.026)nmol·g-1Pr·h-1，（0.156±0.050)μmol·g-1Pr·h-1］，显著减轻A-RI导致的心肌组织结构的损伤。 结论： 地高辛抗血清对A-RI心肌有明显的保护作用，其作用机制可能通过拮抗内洋地黄素，恢复心肌细胞膜Na+-K+-ATP酶活性，减轻细胞内钙超载。     

耗竭糖元对心肌缺血再灌注损伤的保护作用

已知耗竭细胞内糖元以减少缺血心肌组织乳酸堆积可抑制Na/H交换。本实验拟观察这一过程对于缺血后再灌注心肌的影响。采用Wistar大鼠离体等容收缩心脏,实验分两组:对照组(n=5)用K-H液行40分富氧预灌注;实验     

镁对缺血再灌注心肌线粒体合成ATP能力的影响

我们曾经观察到心肌缺血再灌注过程中心肌线粒体Ca~(2+)-ATP酶活性降低,Ca~(2+)大量沉积于线粒体内.含Mg~(2+)溶液灌流对此具有逆转作用。心肌线粒体是心肌细胞进行氧化磷酸化、合成ATP的场所,大量的钙盐沉积,有可能抑制ATP的合成,因此,我们进一步观察含Mg~(2+)溶液灌注对缺血再灌注心肌线粒体合成ATP的影响。实验采用Wistar大鼠离体等容收缩心脏,行Langendorff灌流,常规K-H液预灌流30分钟行低灌流缺血(0.2ml/min,60分钟后,常规K-H液再灌)注30分钟.根据缺血期准注K-H液中Mg~(2+)浓度不同,实验分为0、1.2及15μmol三组(n均为5)。灌流结束后差速离心分离心肌线粒体,加入含有底物ADP     

二氮嗪两种应用对豚鼠缺血再灌注心肌电生理特性的影响

目的：观察二氮嗪两种应用方式对缺血再灌注豚鼠心室乳头肌细胞电生理特性的影响。方法: 24只豚鼠随机分为对照组、实验组、预先给药组(各组8只)，取离体左室乳头肌标本。对照组37 ℃充氧台氏液平衡灌流80 min后，用4 ℃ St.Thommas液灌流停搏30 min，再行充氧台氏液复灌60 min。实验组除4 ℃ St.Thommas液含二氮嗪(100 μmol/L)外余步骤同对照组。预先给药组仅在平衡灌流60 min后改用二氮嗪(100 μmol/L)灌流10 min，冲洗10 min，余步骤同对照组。分别用玻璃微电极技术记录心室乳头肌细胞电生理特性的改变。结果: ⑴ 再灌注5 min、10 min实验组和预先给药组APD50、APD90均明显短于对照组(P<0.01，P<0.05)，而再灌注30 min又明显长于对照组(P<0.01，P<0.05)。⑵ 实验组和预先给药组再灌注30 min动作电位振幅(APA)、超射值(OS)、0期最大除极速度(Vmax) 恢复早于对照组，且复跳时间明显短于对照组(P<0.05)。⑶ 3组停搏后静息电位没有明显差异，预给药组停搏时间长于对照组(P<0.05)。结论: 含二氮嗪的St．Thomas停搏液对缺血再灌注豚鼠心室乳头肌细胞电生理特性的保护效果强于单纯的二氮嗪预先给药和传统的St．Thomas停搏液。     

Na^＋—H^＋交换阻滞剂对离体鼠心缺血后再灌注损伤的保护作用

为评价Na~+-H~+交换阻滞剂对缺血再灌注损伤的保护作用,在改良的Langendoff装置上采取主动脉逆灌方法,用成年Wistar大鼠的离体鼠心完全停灌60min,然后100%再灌注30min造成再灌注损伤模型。阿米洛利(0.2、20、100μmol/L)和呋喃苯胺酸(10μmol/L)为药物保护组及不加药物的对照组。结果显示:阿米洛利和呋喃苯胺酸对再灌注时心肌收缩力、冠脉流量的恢复及抑制再灌注心律失常的发生有明显的作用,阿米洛利抗心律失常的效果呈明显的剂量依赖性。因此提示具Na~+-H~+交换阻断作用的阿米洛利和呋喃苯胺酸具明显的抗再灌注损伤的作用。     

Ryanodine和咖啡因对离体大鼠心脏缺血—再灌注室性心律失常的作用

本工作观察Ryanodine和咖啡因对缺血—再灌性心律失常的作用,并和异搏定的效应进行比较。离体大鼠心脏悬挂于Langendorff灌流架上,用K-H液预灌15min后随机分为5组:模拟缺血组(n=4),持续灌流45min;对照组(n=6)旷置30min,复灌15min;异搏定(n=5)、Ryanodine(n=6)和咖啡因组(n     

含血停搏液镁离子浓度对未成熟心肌保护的影响

目的采用幼兔离体心脏模型,模拟临床上可能出现的含血停搏液Ca2+浓度变化,探讨适宜于未成熟心肌保护的Mg2+浓度.方法3～4周龄长耳白兔,依照含血停搏液不同Mg2+浓度(0.6mmol/L、4.0mmol/L、8.0mmol/L、120mmol/L、16.0mmol/L)随机分为5组,建立Langendorff离体心脏灌注模型.采用Ca2+浓度1.2～1.5 mmol/L的含血停搏液,运用温血停搏液诱导停搏,冷血停搏液间断灌注,低温保护,终末温血停搏液控制性再灌注技术,观察以下指标:1.血流动力学指标:实验前后恢复率:心率、主动脉流量、冠脉流量、心排量、左室收缩压和左室舒张末压;2.心肌含水量;3.冠脉流出液乳酸盐含量;4.心肌肌酸激酶和乳酸脱氢酶漏出率;5.心肌细胞内Na+、Ca2+含量;6.心肌组织ATP含量;7.心肌组织SOD活性、MDA含量;8.心肌超微结构.结果1.心率恢复率、主动脉流量恢复率及左室收缩压恢复率组间总体差异无显著性.而冠脉流量恢复率,心排量恢复率和左室舒张末压恢复率以Mg2+浓度8.0mmol/L和12.0mmol/L为优,0.4mmol/L组最差.2.心肌含水量以Mg2+浓度8.0mmol/L和12.0mmol/L为最低.3.冠脉流出液乳酸盐含量0.4mmol/L组、8.0mmol/L和12.0mmol/L组高于其余2组.4.心肌乳酸脱氢酶漏出率以8.0mmol/L组最低,而肌酸激酶漏出率以8.0mmol/L和12.0mmol/L组为最低.5.心肌细胞内Na+含量以8.0mmol/L和12.0mmol/L组为最低,而心肌细胞内Ca2+含量以8.0mmol/L组量低.6.心肌组织ATP含量以12.0mmol/L组为最高.7.心肌组织SOD活性以8.0mmol/L和12.0 mmol/L组为最高,而MDA含量各组间总体差异无显著性.8.心肌超微结构:8.0mmol/L和12.0mmol/L组表现为基本正常未成熟心肌超微结构,而0.4mmol/L组超微结构有明显损伤表现.结论对于未成熟心肌,当采用温血停搏液诱导停搏,冷血停搏液间断灌注,低温保护,温血停搏液终末控制性再灌注技术时,为避免含血停搏液Ca2+浓度偏高对未成熟心肌的不利影响,应维持含血停搏液中Mg2+浓度在8～12mmol/L.     

丹参滴丸对缺血/再灌注损伤心肌高能磷酸化合物的影响

目的 :进一步探讨丹参滴丸对缺血 /再灌注损伤心肌的保护作用及其机制。方法 :采用Ｌａｎｇｅｎｄｏｒｆｆ离体心脏灌流技术 ,制备心肌缺血 /再灌注损伤模型 ,利用高效液相色谱仪 (ＨＰＬＣ)测定离体大鼠心肌组织中的高能磷酸化合物的含量变化。本实验共分 6组 :正常对照组 :大鼠心脏离体后 ,接在灌流装置上持续灌流 75ｍｉｎ。单纯缺血再灌组 :先预灌 1 5ｍｉｎ ,然后停止灌流 ,保持心脏温度恒定在 37℃ ,在无氧 ,无灌流液的条件下旷置 40ｍｉｎ ,再恢复灌流 2 0ｍｉｎ。丹参滴丸前保护组 :预灌时加丹参滴丸 ,后处理同单纯缺血再灌组。丹参…     

Permeation of NH3/NH4 + and cell pH in colonic crypts of the rat

Colon cells are subjected to high concentrations of NH3 and NH4+, and a sizeable portion of this buffer is absorbed. The flux of these components into cells causes opposite effects on their pH; this effect is largely used to induce an acid load and to observe the mechanism of acid extrusion from cells. We studied cells of microdissected colon crypts loaded with BCECF and superfused with NH4Cl-containing Krebs-Ringer solution. We found a marked transient reduction in pH measured by ratiometric fluorescence microscopy, from a control value of 7.51 +/- 0.041 to 7.15 +/- 0.041 (n = 21), instead of the initial alkalinization found in most cells. This pH was reached at a rate of 0.95 +/- 0.07 pH units/min. Addition of 1 mmol/l furosemide, a blocker of Na+,K+,2Cl- cotransport, to the ammonium solution inverted this acidification toward alkalinization (pH 7.89 +/- 0.041, n = 5), and superfusion with furosemide plus 0.1 mmol/l hexamethylene amiloride, a specific blocker of Na+/H+ exchange, increased this initial alkalinization further to 8.10 +/- 0.117 (n = 7). When Krebs-Ringer with 0 Cl- containing (NH4)2SO4 instead of NH4Cl was superfused, the acid transient was also reverted to alkalinization; however, a higher degree of alkalinization was observed either when 1 mmol/l furosemide was added to the superfusing sulfate solution (when a pH of 7.78 +/- 0.010 was reached), or when ammonium gluconate was used instead of ammonium sulfate. The addition of Ba2+ to the superfusion solution did not alter the initial acidification. These data indicate that in colon crypt cells, basolateral membrane transporters, in particular the Na+,K+,2Cl- cotransporter and the Na+/H+ exchanger (but not Ba(2+)-sensitive K+ channels), mediate the predominant influx of NH4+ ions leading to the initial transient acidification.     

Cardioprotection of SM-15681, an Na+/H+ exchange inhibitor in ischemic and hypoxic isolated perfused rat hearts

We investigated the effects of SM-15681 (N-(aminoiminomethyl)-1-methyl-1H-indole-2-carboxamide monohydrochloride) on Na+/H+ exchange activity in the myocardium and in ischemic and hypoxic injury in isolated perfused rat hearts. These effects were compared with those of ethylisopropyl amiloride (EIPA). Na+/H+ exchange activity was studied with a NH4Cl prepulse technique under HCO3(-)-free conditions. SM-15681 (10(-8)-10(-7) M) inhibited pH recovery of acidosis in the rat myocardium in a concentration-dependent manner and the IC50 value of SM-15681 (80 nM) was similar to that of EIPA. In perfused rat hearts, SM-15681 (10(-6) M) and EIPA (10(-6) M) significantly improved cardiac functions and prevented enzyme release and abnormal elevation of tissue Ca2+ content during 20 min of reperfusion after 40 min of ischemia and 20 min of reoxygenation after 30 min of hypoxia. We conclude that an Na+/H+ exchange inhibitor, SM-15681, shows cardioprotective effects on ischemia/reperfusion and hypoxia/reoxygenation injury. Our results also support the hypothesis that Na+/H+ exchange contributes to the pathophysiology of cardiac ischemic reperfusion injury.     

Na(+)-H+ exchange is not important for pancreatic HCO3- secretion in the pig.

Pancreatic inter- and intralobular duct cells extrude H(+)-ions to interstitial fluid when they secrete HCO3- to pancreatic juice. This study assesses the potential importance of Na(+)-H(+)-ion exchange for H(+)-ion extrusion and secretion of HCO3-, using the Na(+)-H+ exchange blockers amiloride and hexamethylene-amiloride. Intracellular pH (pHi) in inter- and intralobular pancreatic duct epithelium was measured using BCECF fluorescence. H(+)-ion efflux was measured using a NH4Cl prepulse, acid-loading technique. In HCO3(-)-free media, pHi recovery following acid loading was blocked by amiloride (10(-4) M) and hexamethylene-amiloride (10(-6) M), demonstrating amiloride- and hexamethylene-amiloride-sensitive Na(+)-H+ exchange. However, 5 x 10(-6) M hexamethylene-amiloride did not reduce secretin-dependent pancreatic HCO3- secretion in vivo. Maximal H(+)-efflux through Na(+)-H+ exchange was 1.5 +/- 0.2 mumol min-1 ml cell volume-1, i.e. less than 1% of estimated net H(+)-ion efflux during HCO3- secretion. Conclusion: amiloride- and hexamethylene amiloride sensitive Na(+)-H+ exchange is not important for secretin-dependent pancreatic HCO3- secretion in the pig. Other mechanisms for H+ extrusion dominate.     

Contribution of Na+/H+ exchange to pH regulation in pulmonary artery smooth muscle cells.

In blood vessels in the systemic circulation, the plasmalemmal Na+/H+ exchanger has been implicated in a variety of cellular functions, including the regulation of intracellular pH (pHi) and cell volume, and the response to smooth muscle mitogens. The role of this transport system in pulmonary vascular smooth muscle has not been explored. The present study examined the characteristics of Na+/H+ exchange in cultured guinea pig pulmonary artery smooth muscle cells. These cells were subjected to an acid load, and the recovery from acid loading was monitored using the fluorescent pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). In the absence of HCO3-, pHi recovery from acid loading was dependent on external Na+ and was inhibited by the Na+/H+ exchange inhibitor dimethylamiloride (DMA) (recovery rate was reduced from 54.4 +/- 5.5 to 12.8 +/- 2.0 mmol H+/liter.min). This exchanger was also active in the presence of HCO3-; DMA reduced resting pHi and slowed the rate of recovery from acid loading in HCO3- buffers. However, in the presence of HCO3-, other transport systems, presumably HCO3-/Cl- exchange, also contribute to the regulation of pHi. In HCO3- buffers, the rate of recovery from acid load averaged 40.8 +/- 1.8 mmol H+/liter.min. Addition of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), an inhibitor of HCO3-/Cl- exchange, slowed this recovery to 25.5 +/- 1.6 mmol H+/liter.min. A combination of DIDS and DMA further slowed the recovery to 19.7 +/- 1.5 mmol H+/liter.min. These findings indicate that the Na+/H+ exchanger plays a significant role in the regulation of pHi in pulmonary artery smooth muscle cells, even in HCO(3-)-containing buffers.     

Sodium transport in the freshwater mussel, Carunculina texasensis (Lea)

Freshwater bivalves maintain a Na steady state in artificial pondwater: JiNa = 1.2 +/- 0.1 mumol/g dry tissue per h. Na uptake is Cl independent. The affinity (KS) of the Na transport system is 0.15-0.23 mmol Na/1. Sodium influx is coupled to H and/or NH4 exchange. Salt depletion stimulates JiNa 300% relative to nondepleted animals with no change in Ks. Injected ammonium ion stimulates JiNa. Sodium transport is inhibited 84% by 0.5 mM amiloride but is not affected by 4 mM NH4 or 1 mM furosemide in the bathing solution or injection of acetazolamide (0.26 mumol/ml blood).     

Role of sodium in mediator release from human basophils.

We studied the effect of extracellular sodium concentration on histamine release (HR) from human basophils initiated by immunologic and nonimmunologic stimuli. We found that lowering extracellular sodium markedly enhances HR induced by an immunologic stimulus from these cells. In buffer in which sodium had been replaced with univalent ions of strong bases, enhancement of HR increased as extracellular sodium decreased. Enhancement was the result of increased duration of release. When sucrose was used for replacement of sodium, we also observed that enhancement of HR increased as extracellular sodium decreased, but there was some lessening of enhancement at [Na+]e between 5 and 10 mmol/L. Ouabain, which is an inhibitor of the Na+/K+ adenosine triphosphatase, and bumetanide and furosemide, which are inhibitors of Cl(-)-dependent Na(+)-K+ cotransport, caused small increases in enhancement of HR by sodium-deficient buffers; 4,4'-diisothiocyanostilbene-2-2'-disulfonic acid, an anion transport inhibitor, caused some inhibition of enhancement of HR. Analogues of amiloride, such as 5-(N-N-hexamethylene) amiloride (HMA) and 5-(N-4-chlorobenzyl)-2'-4'dimethylbenzamil (CBDMB), inhibit Na+/H+ exchange, Na+/Ca++ exchange, and Na+ channels. Interestingly, at higher doses, HMA and CBDMB caused marked enhancement of HR in both normal and sodium-deficient buffers. These results suggest that several cellular regulatory mechanisms potentially are important for normal basophil secretion. The most likely are pH regulatory mechanisms that include Na+/H+ exchange and anion exchangers that transport alkaline equivalents. Our findings enhancement of basophil HR by HMA and CBDMB is particularly noteworthy in light of the recent interest in use of amiloride by inhalation for therapy of lung disease in patients with cystic fibrosis.     

Regulation of the Na+2Cl–K+ cotransporter in isolated rat colon crypts

The Na(+2)Cl(-)K+ cotransporter accepts NH4+ at its K+-binding site. Therefore, the rate of cytosolic acidification after NH4+ addition to the bath (20 mmol/l) measured by BCECF fluorescence can be used to quantify the rate of this cotransporter. In isolated colon crypts of rat distal colon (RCC) addition of NH4+ led to an initial alkalinization, corresponding to NH3 uptake. This was followed by an acidification, corresponding to NH4+ uptake. The rate of this uptake was quantified by exponential curve fitting and is given in arbitrary units (delta fluorescence ratio units/1000 s). In pilot experiments it was shown that the pH signal caused by the Na(+)2Cl(-)K+ co-transporter could be amplified if the experiments were carried out in the presence of bath Ba2+ to inhibit NH4+ uptake via K+ channels. Therefore all subsequent experiments were performed in the presence of 1 mmol/l Ba2+. In the absence of any secretagogue, preincubation of RCC in a low-Cl- solution (4 mmol/l) for 10 min enhanced the uptake rate significantly from 1.70+/-0.11 to 2.54+/-0.27 U/1000 s (n=20). The addition of 100 mmol/l mannitol (hypertonic solution) enhanced the rate significantly from 1.93+/-0.17 to 2.84+/-0.43 U/1000 s (n=5). Stimulation of NaCl secretion by a solution containing 100 micromol/l carbachol (CCH) led to a small but significant increase in NH4+ uptake rate from 2.06+/-0.34 to 2.40+/-0.30 U/1000 s (n= 11). The increase in uptake rate observed with stimulation of the cAMP pathway by isobutylmethylxanthine (IBMX) and forskolin (100 micromol/l and 5 micromol/l, respectively) was from 2.39+/-0.24 to 3.06+/-0.36 U/1000 s (n=24). Whatever the mechanism used to increase the NH4+ uptake rate, azosemide (500 micromol/l) always reduced this rate to control values. Hence three manoeuvres enhanced loop-diuretic-inhibitable uptake rates of the Na(+)2Cl(-)K+ cotransporter: (1) lowering of cytosolic Cl- concentration; (2) cell shrinkage; (3) activation of NaCl secretion by carbachol and (4) activation of NaCl secretion by cAMP. The common denominator of all four activation pathways may be a transient fall in cell volume.     

A role for Na+/H+ exchange in pH regulation in Helix neurones

We have used the pH-sensitive fluorescent dye 8-hydroxypyrene-1,3,6-trisulphonic acid (HPTS) to reexamine the mechanisms that extrude acid from voltage-clamped Helix aspersa neurones. Intracellular acid loads were imposed by three different methods: application of weak acid, depolarization and removal of extracellular sodium. In nominally CO2/HCO3-free Ringer the rate of recovery from acid loads was significantly slowed by the potent Na+/H+ exchange inhibitor 5-[N-ethyl-N-isopropyl]-amiloride (EIPA, 50 microM). Following depolarization-induced acidifications the rate of intracellular pH (pHi) recovery was significantly reduced from 0.41 +/- 0.13 pH units.h-1 in controls to 0.12 +/- 0.09 pH units.h-1 after treatment with EIPA at pHi approximately equal to 7.3 (n = 7). The amiloride analogue also reduced the rate of acid loading seen during extracellular sodium removal both in the presence and absence of the Na(+)-dependent Cl-/HCO3- exchange inhibitor 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulphonic acid (SITS, 50 microM). This is consistent with EIPA inhibiting reverse-mode Na+/H+ exchange. In 2.5% CO2/20 mM HCO3-buffered Ringer pHi recovery was significantly inhibited by SITS, but unaffected by EIPA. Our results indicate that there are two separate Na(+)-dependent mechanisms involved in the maintenance of pHi in Helix neurones: Na(+)-dependent Cl-/HCO3- exchange and Na+/H+ exchange. Acid extrusion from Helix neurones is predominantly dependent upon the activity of Na(+)-dependent Cl-/HCO3- exchange with a lesser role for Na+/H+ exchange. This adds further weight to the belief that the Na+/H+ exchanger is ubiquitous.     

Factors affecting ammonium uptake by C11 clone of MDCK cells

In several tissues ammonium ions are able to use the transport pathways of other ions, particularly of K+. We investigated this possibility in the C11 clone of MDCK cells, thought to represent intercalated cells, in control and 0 Cl- conditions. Cell pH was measured by ratiometric fluorescence microscopy using the pH indicator BCECF. After preincubating the cells for 10 min in control or 0 Cl- (substituted by gluconate) Ringer, an ammonium pulse was applied to induce cell acidification. The magnitude of the initial alkalinization (DeltapH) was 0.24+/-0.03 ( n=28) pH units in controls, which fell to 0.023+/-0.01 ( n=12) in 0 Cl-, suggesting uptake of NH4+ balancing the alkalinization by NH3. Addition of 10(-3) M bumetanide or furosemide to the 0 Cl- medium, or 10(-4 )M hexamethylene amiloride, did not alter DeltapH. However, with 5 mM Ba+, DeltapH increased to 38% of control. When 2.5x10(-4) M ouabain, an inhibitor of Na+-K+ ATPase, was used, DeltapH increased to 46% of control. Inhibition of H+-K+ ATPase by SCH28080 or by omeprazol caused significant increase in DeltapH. In 0 Cl- solution, these cells underwent a mean volume reduction (-d V) of -10.24+/-1.96% per 10 min as measured by confocal microscopy. To investigate if NH4+ influx was regulated by cell volume or by cell Cl-, volume reduction was avoided by two procedures. When preincubating with NPPB, a Cl- channel blocker, in 0 Cl-, volume reduction was inhibited (d V=-2.12% per 10 min), and DeltapH was 0.24+/-0.04 ( n=5). When the cells were preincubated in hypotonic 0 Cl- (260 mosmol/l), cell volume reduction was abolished (d V=+2.6% per 10 min) and DeltapH was 0.52+/-0.07 ( n=7). Thus, activation of NH4+ influx by several transporters was due to volume reduction rather than to [Cl-] alteration.