Sodium-sensitive, calcium-independent potassium conductance in the leech giant glial cell

 We have measured membrane current, membrane potential and intracellular Na⁺ and Ca²⁺ concentrations, [Na⁺]_i and [Ca²⁺]_i, of the giant glial cell in the nervous system of the leech Hirudo medicinalis using conventional microelectrodes and the fluorescent dyes sodium-binding benzofuran isophthalate (SBFI) and fura-2. When the Na⁺ was removed from the saline, the membrane conductance increased twofold from 1.29±0.1 μS to 2.57±0.18 μS (mean ± SEM; n=27). The rise in membrane conductance was accompanied by a current, which reversed around –74 mV, and the amplitude of K⁺-induced depolarizations or currents increased during Na⁺ removal, suggesting an increase in the K⁺ conductance of the glial membrane. We also monitored [Ca²⁺]_i when removing external Na⁺ in the presence and absence of external Ca²⁺, and during injection of the Ca²⁺-chelator BAPTA into the cells. Our results indicate that Na⁺ modulates a K⁺ conductance of these glial cells, independent of intra- and extracellular Ca²⁺. Received: 1 April 1998 / Received after revision and accepted: 22 May 1998     

马桑内酯致痫大鼠海马神经细胞外Na+、K+活度的变化

目的和方法：应用Na⁺、K⁺选择性微电极检测马桑内酯致痫大鼠海马及海马脑片神经细胞外Na⁺、K⁺活度的改变。结果：海马内注射马桑内酯（5 μL,5×10^-4 mol/L）致痫大鼠30 s、1 min和2 min后,海马神经细胞外Na⁺活度分别低于对照组27.7 mmol/L、50.3 mmol/L和57.8 mmol/L,而K活度则分别高于对照组2.3 mmol/L、2.4 mmol/L和2.9 mmol/L（P<0.01）。3 min后,K⁺活度基本恢复至对照水平,而Na⁺活度仍持续低于对照水平（P<0.01）。海马脑片的实验结果与在体实验相似。结论：海马神经细胞处于癫痫状态时,存在Na⁺内流、K⁺外流现象。     

参麦注射液对大鼠急性心肌缺血再灌注损伤的影响

目的:观察参麦注射液对大鼠急性心肌缺血再灌注损伤的影响,并探讨其作用机制。方法:结扎冠状动脉左前降支10min再灌15min复制大鼠急性心肌缺血再灌注损伤模型,描记标准肢体Ⅱ导联心电图,测定心肌组织匀浆中超氧化物歧化酶(SOD)、Na⁺,K⁺-ATP酶和Ca²⁺-ATP酶活性及丙二醛(MDA)含量,电镜观察心肌线粒体改变。结果:参麦注射液使再灌注性心律失常的发生率低于模型组、持续时间较短,心肌组织匀浆中SOD、Na⁺,K⁺-ATP酶和Ca²⁺-ATP酶的活性高于模型组,MDA的含量低于模型组,心肌线粒体损伤轻于模型组。结论:参麦注射液对大鼠急性心肌缺血再灌注损伤有明显的防治作用,其机制与减轻氧自由基及钙超载损伤有关。     

缺血预处理快速效应对兔急性缺血脊髓的保护作用

目的:探讨缺血预处理快速相对兔腹主动脉短暂阻断致缺血脊髓的保护作用。方法:36只雄性新西兰兔随机分成3组(n=12):即缺血再灌注损伤组(IR组)、缺血预处理组(IPC+IR组)及假手术组(Sham组)。IR组阻闭兔腹主动脉肾下段20min,复制兔脊髓缺血损伤模型;IPC+IR组预先阻闭腹主动脉肾下段6min,再灌注30min后再次阻闭腹主动脉肾下段20min;Sham组除不夹闭腹主动脉外,其余处理同IR组。再灌注后8h、12h、24h和48h分别对动物神经功能评分,然后,处死动物取脊髓(L_5-7),分别行组织病理学观察及测定脊髓组织中Na⁺,K⁺-ATP酶的活性。结果:Sham组及IPC+IR组神经功能评分各时点均明显高于IR组(P＜0.01);Sham组及IPC+IR组脊髓前角正常神经细胞数明显多于IR组(P＜0.01);Sham组及IPC+IR组脊髓组织中Na⁺,K⁺-ATP酶的活性明显高于IR组(P＜0.01)。结论:缺血预处理快速相对兔急性缺血脊髓有显著的保护作用,这种保护作用可能与稳定Na⁺,K⁺-ATP酶的活性有关。     

Evidence for the presence of a Na+-H+ exchanger in the endolymphatic sac epithelium of guinea-pigs

 The intracellular pH (pH_i) of epithelial cells from the endolymphatic sac (ES) of the guinea-pig was measured microfluorometrically with the pH-sensitive fluorescent dye, 2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) to examine the presence of a Na⁺-H⁺ exchanger (NHE) in the ES epithelial cells. pH_i recovery from acid loading with an NH₄ ⁺-prepulse in a nominally HCO₃-free solution was dependent on extracellular Na⁺ ([Na⁺]_o) and was inhibited by amiloride and its analogue ethylisopropylamiloride (EIPA), suggesting that a decreased pH_i induced by an acute acid load may be equilibrated by a NHE. In the steady-state, amiloride had no effect on pH_i, indicating that the NHE activity is low at the resting pH_i. However, the intracellular acidification induced by the removal of [Na⁺]_o was inhibited by the simultaneous application of amiloride. H⁺-efflux rate (J _H, mean activity of NHE), which was calculated as the product of the recovery rate (dpH_i/dt) from the acid loading and the intrinsic buffering capacity (β_i) at the corresponding pH_i, was decreased as pH_i was increased. The concentration/response curve for the inhibition of initial J _H by EIPA revealed an apparent 50% inhibitory constant (K _i ) of 0.85 μM. Kinetic analysis of initial J _H as a function of [Na⁺]_o revealed a Michaelis-Menten constant (K _m) of 24.14 mM for Na⁺-dependent H⁺ efflux. The results indicate that NHE in the ES epithelium belongs to an amiloride-sensitive subtype. Received: 5 August 1997 / Received after revision and accepted: 26 February 1998     

Effect of extracellular pH on the myo-inositol transporter SMIT expressed in Xenopus oocytes

 The myo-inositol transporter SMIT is expressed particularly at high extracellular osmolarity and serves to accumulate the osmolyte myo-inositol. Transport of myo-inositol is coupled to the cotransport of Na⁺ and is electrogenic. In Xenopus oocytes injected with mRNA encoding SMIT but not in water-injected oocytes, myo-inositol creates an inward current that is dependent on the ambient Na⁺ concentration. The present study has been performed to elucidate the pH dependence of myo-inositol-induced currents. Therefore, Xenopus oocytes were injected with mRNA encoding SMIT and two-electrode voltage-clamp studies were performed. The myo-inositol-induced currents in oocytes expressing SMIT were found to have a sigmoidal dependence on the ambient pH between pH 5.5 and 8.5 with an apparent K _i of 0.21±001 μM H⁺ and a Hill coefficient of 1.80±0.16. Kinetic analysis of the myo-inositol-induced currents at pH 8.0 and –90 mV holding potential revealed a Hill coefficient of 0.93±0.07 and an apparent K _m for myo-inositol of 0.031±0.003 mM as well as a Hill coefficient of 1.64±0.24 and an apparent K _m of 38.8±4.1 mM for Na⁺. A decrease of the Na⁺ concentra-tion from 150 mM to 50 mM significantly altered the maximal observed current and increased the apparent K _m for myo-inositol. Acidification to pH 6.5 significantly increased the apparent K _m for myo-inositol and for Na⁺ to 0.057±0.005 mM and 73.9±4.8 mM, respectively. The Hill coefficients for myo-inositol and Na⁺ were not affected and remained close to 1 for myo-inositol and 2 for Na⁺. In summary, acidification impedes SMIT-mediated myo-inositol transport at least partially by decreasing the affinity of the carrier for Na⁺. The impaired Na⁺ binding subsequently decreases binding and transport of myo-inositol. Received: 20 April 1998 / Received after revision: 29 June 1998 / Accepted: 13 July 1998     

应用酵母双杂交技术筛选与RANK蛋白新基序IVVY相互作用的蛋白

目的: 应用酵母双杂交技术筛选小鼠巨噬细胞中与核因子κB受体激活因子(RANK)蛋白上新基序IVVY相互作用的蛋白,以探寻RANKL/RANK系统中介导破骨细胞形成的RANK下游新信号转导蛋白。方法: 应用GAL4酵母双杂交系统3,构建仅包含编码新基序⁵³⁵IVVY⁵³⁸ 的一小段RANK的cDNA片段为诱饵质粒pGBKT7-IVVY,并与巨噬细胞cDNA文库质粒pGADT7-library共转化AH109酵母,筛选与RANK蛋白新基序IVVY相互作用的蛋白,通过回复性杂交实验验证其可靠性,并对阳性克隆进行测序和基因同源性分析。结果: 筛选出4个可能与IVVY基序有相互作用的蛋白,包括Ring1和YY1结合蛋白(RYBP)、ATP结合盒、E2F转录因子和热休克蛋白8,其中表达RYBP的阳性克隆出现频率高、速度快。结论: 应用酵母双杂交技术成功地筛选出4个可能与IVVY基序相互作用的候选蛋白,其中RYBP可能性最大。     

人肝再生增强因子cDNA的克隆及其酵母双杂交载体的构建

目的：获取人肝再生增强因子（hALR)阅读框的cDNA及构建其酵母双杂交系统的“诱饵”质粒。方法：利用RT－PCR方法，从人胎肝组织中扩增出一约380bp的DNA片段，重组入pGBKT7载体中，构建成pGBKT7-hALR，然后研究此重组质粒在酵母AH109中的表达情况并用于筛选人肝cDNA文库，结果：获得的378bp的DNA序列与文献报道的人ALR序列一致；转化的酵母细菌在选择性培养基SD／-Trp上培养65小时，长出约Φ1mm大小的白色菌落，而在SD／－Trp/-His上不生长；酵母提取液的Western blot分析，证实ALR基因在AH109以融合蛋白的形式表达，并具有免疫活性；初步得到hALR相互作用的阳性克隆。结论：pGBKT7-hALR对宿主菌AH109没有毒性作用，也没有自身激活报告基因，可作为酵母双杂交系统中的“诱饵”质粒。     

Ionic currents expressed in a cell line derived from the organ of Corti of the Immortomouse

 We have investigated the maturation of adult hair cell electrophysiology in a population of precursor cells in a conditionally immortal cell line. The cell line, UB/OC-2, from the embryonic organ of Corti of the H-2Kb-tsA58 transgenic mouse, permits cells to grow proliferatively at 33°C and to differentiate at 39°C. Whole-cell patch-clamp recordings showed that proliferating cells had a different electrophysiology to differentiating cells. Differentiating cells had a conditionally expressed slowly activating inward current activated by hyperpolarization. The current was not blocked by extracellular application of 0.5 mM Ba²⁺, but was blocked reversibly by 2 mM Cs⁺. The current was found to be carried by both K⁺ and Na⁺ ions (P _K/P _Na=2.2) and activated by 10 μM forskolin. These properties identify the slowly activating current as I _h. A proportion of proliferating and differentiating cells exhibited a voltage-gated Na⁺ current, I _Na. I _Na was abolished in Na-free external medium and was inhibited reversibly by tetrodotoxin (TTX) with K _i=64 nM. Together these results suggest that proliferating and differentiating hair cell precursors in the immortal cochlear cell line UB/OC-2 express currents which are also found in developing hair cells. Received: 6 January 1999 / Received after revision: 1 February 1999 / Accepted: 2 February 1999     

应用酵母双杂交技术筛选干扰素β结合蛋白的研究

目的 应用酵母双杂交技术筛选干扰素β（IFNp）结合蛋白。方法 用多聚酶链反应（PCR）技术扩增IFNB基因，连接酵母表达载体pGBKT7构建诱饵质粒，转化酵母细胞AH109，与含人肝细胞cDNA文库质粒的酵母细胞Y187进行配合，在营养缺陷型培养基（SD／-Trp-Leu-His-Ade）和X-α-半乳糖（X-α-gal列）上进行双重筛选阳性克隆，提取酵母质粒后转化大肠埃希菌（DH5α）并经氨苄西林抗性筛选，提取单克隆菌落质粒，进行酶切鉴定及序列测定，并进行生物信息学分析。结果 应用酵母双杂交技术筛选出阳性克隆29个，经生物信息学分析，排除读码框架不正确者，最后得到19个克隆基因，编码14种已知蛋白和一种未知功能蛋白。初步发现铁蛋白与IFNB具有结合作用。结论 应用酵母双杂交技术筛选出多种与IFNB具有结合作用的蛋白。     

应用酵母双杂交系统筛选E2F1相互作用蛋白

目的:应用酵母双杂交系统从人正常胃黏膜细胞的cDNA文库中筛选E2F1转录因子的相互作用蛋白。方法:以pGBKT7-E2F1为诱饵质粒筛选人正常胃黏膜细胞的cDNA文库,得到阳性克隆,反复验证,并对验证后的阳性克隆进行测序及同源性分析。结果:从人正常胃黏膜细胞的cDNA文库中筛选得到20个阳性克隆,经测序和同源性分析,筛除假阳性克隆,最终得到18个不同的候选基因序列。其中,17个为可知基因序列,它们分别是:组织蛋白酶B、SIVA1、干扰素调节因子7、鸟苷酸激酶1、ATP酶抑制因子1、核糖体蛋白SA、纤溶酶原激活物抑制剂1 mRNA结合蛋白、精氨琥珀酸合酶1、卵泡抑素类似物3、金属硫蛋白2A、内质网钙结合蛋白1、WNT1可诱导信号通路蛋白2、CDC42 效应蛋白1、可溶性半乳糖凝集素结合蛋白1、中胚层发育候选2、外切酶体成分7和含EGF腓骨蛋白样胞外基质蛋白 1,尚有一未知基因片段。结论:应用酵母双杂交系统成功筛选出18种E2F1相互作用蛋白,为进一步探讨E2F1影响胃癌细胞生物学行为的分子机制奠定了基础。     

On the functional interaction between nicotinic acetylcholine receptor and Na+,K+-ATPase

Previous studies have shown that nanomolar acetylcholine (ACh) produces a 2 to 4-mV hyperpolarization of skeletal muscle fibers putatively due to Na⁺,K⁺-ATPase activation. The present study elucidates the involvement of the nicotinic ACh receptor (nAChR) and of Na⁺,K⁺-ATPase isoform(s) in ACh-induced hyperpolarization of rat diaphragm muscle fibers. A variety of ligands of specific binding sites of nAChR and Na⁺,K⁺-ATPase were used. Dose–response curves for ouabain, a specific Na⁺,K⁺-ATPase inhibitor, were obtained to ascertain which Na⁺,K⁺-ATPase isoform(s) is involved. The ACh dose–response relationship for the hyperpolarization was also determined. The functional relationship between these two proteins was also studied in a less complex system, a membrane preparation from Torpedo electric organ. The possibility of a direct ACh effect on Na⁺,K⁺-ATPase was studied in purified lamb kidney Na⁺,K⁺-ATPase and in rat red blood cells, systems where no nAChR is present. The results indicate that binding of nAChR agonists to their specific sites results in modulation of ouabain-sensitive (most probably α2) isoform of Na⁺,K⁺-ATPase, leading to muscle membrane hyperpolarization. In the Torpedo preparation, ouabain modulates dansyl-C6-choline binding to nAChR, and vice versa. These results provide the first evidence of a functional interaction between nAChR and Na⁺,K⁺-ATPase. Possible interaction mechanisms are discussed.     

High salt diet down-regulates proximal tubule Na+, K+-ATPase activity in Dahl salt-resistant but not in Dahl salt-sensitive rats: evidence of defective dopamine regulation

Nishi , A., Bertorello , A. M. & Aperia , A. 1992. High salt diet down-regulates proximal tubule Na⁺, K⁺-ATPase activity in Dahl salt-resistant but not in Dahl salt-sensitive rats; evidence of defective dopamine regulation. Acta Ptiysiol Scand 144 , 263–267. Received 26 July 1991, accepted 25 October 1991. ISSN 0001–6772. Department of Paediatrics, Karolinska Institute, Sweden We examined the regulation of Na⁺, K⁺-ATPase activity in proximal tubule segments during a high salt diet in prehypertensive Dahl salt-sensitive and salt-resistant rats. Rats were placed on normal salt or high salt diets (0.9% saline as drinking water). During the normal salt diet, Na⁺, K⁺-ATPase activity was not different between Dahl salt-sensitive and salt-resistant rats. After 2 days and 10 days on a high salt diet, Na⁺, K⁺-ATPase activity in Dahl salt-resistant rats significantly decreased when compared to Dahl salt-resistant rats on a normal salt diet (P < 0.01). The decreased Na⁺, K⁺-ATPase activity in Dahl salt-resistant rats during a high salt diet was reversed by treatment with an inhibitor of aromatic l -amino acid decarboxylase (dopamine synthesizing enzyme), benserazide. In contrast, Na⁺, K⁺-ATPase activity did not decrease during the high salt diet and benserazide had no effect on Na⁺, K⁺-ATPase activity in Dahl salt-sensitive rats. These results indicate that Dahl salt-sensitive rats do not have the capacity to down-regulate the proximal tubule Na⁺, K⁺-ATPase activity during a high salt diet. Indirect evidence suggests that the regulation of Na⁺, K⁺-ATPase activity by locally produced dopamine is absent in Dahl salt-sensitive rats.     

Calcium supplementation and thyroid hormone protect against gentamicin-induced inhibition of proximal tubular Na+, K+-ATPase activity and other renal functional changes

Gentamicin can cause proximal tubule necrosis. We have shown that inhibition of PT Na⁺, K⁺-ATPase activity is rapidly induced by gentamicin. We have now investigated whether manipulations known to attenuate the negative effects of gentamicin on renal excretory capacity, i.e. high calcium intake and L-thyroxine treatment, will also attenuate gentamicin-induced inhibition of Na⁺, K⁺-ATPase activity and ameliorated signs of proximal tubule damage. Rats were gentamicin- or vehicle-treated for 7 days. Sub-groups were given 4% calcium (Ca) supplements or L-thyroxine 20 μg 100 g^-1 body weight daily. Gentamicin significantly reduced the glomerular filtration rate and increased the urinary excretion of the proximal tubule lysosomal enzyme, N-acetyl-β-d -glucosaminidase. Gentamicin significantly reduced proximal tubule Na⁺, K⁺-ATPase activity, measured in single permeabilized proximal tubule segments. Sodium excretion was inversely correlated to proximal tubule Na⁺, K⁺-ATPase activity. Both calcium and L-thyroxine alleviated all gentamicin-induced side-effects on renal function as well as on proximal tubule Na⁺, K⁺-ATPase activity. Calcium and L-thyroxine had no significant effect on renal function. L-thyroxine, but not calcium, increased proximal tubule Na⁺, K⁺-ATPase activity in control rats. Renal cortical tissue gentamicin concentration was not influenced by calcium but was significantly lowered by L-thyroxine. Two procedures which, via different mechanisms, afford protection from gentamicin-induced changes in renal function also give protection from gentamicin-induced inhibition of Na⁺, K⁺-ATPase activity. This suggests that loss of integrity of the Na⁺, K⁺-ATPase enzyme contributes to gentamicin-induced nephrotoxicity.     

Renal Na+, K+-ATPase in Dahl salt-sensitive rats: K+ dependence,effect of cell environment and protein kinases

Na⁺, K⁺-ATPase in renal epithelial cells plays an important role in the regulation of Na⁺ balance, extracellular volume and blood pressure. The function of renal Na⁺, K⁺-ATPase in Dahl salt-sensitive (DS) rats, an animal model for salt-sensitive hypertension, and Dahl salt-resistant (DR) rats has been studied. In Na⁺, K⁺-ATPase partially purified from renal cortex, affinities and the Hill coefficients for Na⁺ and K⁺ activation were similar in DS and DR rats. Only one component of low ouabain affinity site was found in both strains, indicating the presence of the al isoform. Protein kinase C and cAMP-dependent protein kinase phosphorylated Na⁺, K⁺-ATPase α subunit in DS and DR rats, and the phosphorylation by protein kinase C was associated with an inhibition of enzyme activity. The kinetic parameters for K⁺ activation were also studied in a preparation of basolateral membranes and were found to be similar in DS and DR rats. In a preparation of cortical tubule cells, Na⁺, K⁺-ATPase activity was determined as ouabain-sensitive oxygen consumption (OS Qo₂). Maximal OS Qo₂, measured in Na⁺ loaded cells, was the same in DS and DR rats. The K₀₆ for K⁺ was significantly lower in DS than DR rats (0.163 ±0.042 vs. 0.447 + 0.061 mM, P < 0.05), indicating that factors regulating Na⁺, K⁺-ATPase activity in intact cells are altered in DS rats. Kinetic parameters for Na⁺ activation in cells were the same in both strains. In summary, the function of renal Na⁺, K⁺-ATPase molecule is not altered in DS rats. The intracellular systems that regulate renal Na⁺, K⁺-ATPase activity might be different in DS and DR rats.     

Sodium-dependent regulation of sodium,potassium-adenosine-tri-phosphatase (Na+, K+-ATPase) activity in medullary thick ascending limb of Henle segments. Effect of cyclic-adenosine-monophosphate guanosine-nucleotide-binding-protein activity and arginine vasopressin

This study examine the regulation Na⁺, K⁺-ATPase activity in the medullary thick ascending limb of Henle Na⁺, K⁺-ATPase activity was determined in medullary thick ascending limb of Henle (mtal) segments dissected from rat kidneys. The sodium concentration in the medium (Na,) was 20 or 70 mM. Since the segments were permeabilized, intracellular Na⁺ (Na,) was assumed to be the same as Na₂. Dibuturyl cyclic adenosine monophosphate (dbcAMP) and forskolin inhibited Na⁺, K⁺-ATPase activity independently of Na_m. Arginine vasopressin (AVP) receptors coupled to adenylate cyclase have been identified in the medullary thick ascending limb of Henle. At Na_m= 20 mMAVP caused a dose-dependent inhibition of Na⁺, K⁺-ATPase activity with a maximal effect (49%) at 10^-8 m. This inhibition was abolished in the presence of the adenylate cyclase inhibitor 2,5-dideoxyadenosine (2, 5-DDA). AVP had no effect on Na⁺, K⁺-ATPase activity in the mTAL at Na_m= 70 mM. The guanosine-diphosphate analogue GDPβS inhibited Na⁺, K⁺-ATPase activity at Na_m= 70 mM but not at Na_m= 20 mM. We conclude that increased cyclic adenosine monophosphate (CAMP) levels inhibit Na⁺, K⁺, ATPase activity in mTAL. AVP can, depending on Na₂ produce this effect by adenylate cyclase activation. The guanonine nucleotide binding protein G-protein might be the site of Na⁺-dependence.     

Stimulation of Na+, K+-pump activity in skeletal muscle by methylxanthines: evidence and proposed mechanisms

Evidence is presented to support the hypothesis that submillimolar concentrations of methylxanthines stimulate Na⁺, K⁺-ATPase activity in skeletal muscle. Administration of methylxanthines to skeletal muscle results in plasma membrane hyperpolarization and increased rates of K⁺ uptake and Na⁺ efflux. These effects are both dose- and time-dependent and inhibited by blockers of the Na⁺, K⁺ ATPase. The mechanisms for stimulation of Na⁺, K⁺-ATPase activity and the signal transduction pathways are not known. The methylxanthine concentrations required for stimulation of Na⁺, K⁺-ATPase activity are less than those required to cause a 50% inhibition of phosphodiesterase activity, and therefore increases in cyclic AMP due to inhibition of the enzyme are not involved. Possible mechanisms by which methylxanthines may increase Na⁺, K⁺-ATPase activity include: (1) a role for increased intracellular [Ca²⁺]; (2) Ca²⁺ or adenosine-receptor-mediated increases in intracellular cyclic AMP; and (3) a direct action of methylxanthines on the Na⁺, K⁺ ATPase.     

Isoform of Na+, K+-ATPase from rumen epithelium identified and quantified by immunochemical methods

Using biopsies of rumen epithelium papillae a net influx of [⁸⁶Rb⁺] was measured corresponding to a high concentration of Na⁺, K⁺-pumps found in [³H]ouabain-binding studies ( 18 ). In the present study the Na⁺, K⁺-ATPase in papillae homogenates is compared with purified (Na⁺, K⁺)-ATPase from different sources, immunochemically characterized with respect to the isoform of the hydrolytic α subunit and the concentration of pumps substantiated by a novel immunochemical method. Na⁺, K⁺-ATPase purified from bovine kidney was shown to contain one homogeneous high-affinity population of [³H]ouabain-binding sites (K_d 1.37 n M ). The ouabain-binding capacity was 0.82 nmol (mg protein)^?1. Site-directed polyclonal antibodies raised to isoform-specific sequences of the three known α-subunit isoforms and monoclonal α₁-specific antibodies were used for isoform characterization on western blots of peptides separated by SDS-polyacrylamide gel electrophoresis. All three isoforms were present in Na⁺, K⁺-ATPase prepared from bovine brain. The α isoform of bovine kidney Na⁺, K⁺-ATPase and of rumen epithelium homogenate appeared to be α₁ whereas α₂ and α₃ were undetectable. Using an α₁-specific antibody and ¹²⁵I-labelled antimouse IgG the content of (Na⁺, K⁺)-ATPase in rumen epithelium was determined by comparison of the signal from known amount of bovine kidney Na⁺, K⁺-ATPase on western blots. By this method rumen epithelium was found to contain 2.6 nmol Na⁺, K⁺-ATPase (g wet wt)^?1, i.e. a similarly high or even higher concentration than previously seen in ouabain-binding studies on biopsies.     

Forskolin–induced down–regulation of Na+,K+–ATPase activity is not associated with internalization of the enzyme

Activation by protein kinase A by forskolin phosphorylates and inactivates Na⁺,K⁺-ATPase in COS-7 cells (Cheng et al. 1997b). In this study we show, using [³H]ouabain binding, that forskolin-induced inhibition of Na⁺,K⁺-ATPase activity is not because of internalization of the enzyme. The effect of forskolin on Na⁺,K⁺-ATPase activity was examined by two independent methods, ouabain-sensitive ⁸⁶Rb⁺ uptake in intact cells and ATP hydrolysis in microsomal preparations from cells. The change in number of functional pumps on cell surface before and after protein kinase A activation was assessed by [³H]ouabain binding measured under equilibrium conditions. Cells, which had been ATP-depleted by antimycin A and 2-deoxyglucose treatment, served as a positive control for the internalization of Na⁺,K⁺-ATPase. Activation of protein kinase A with forskolin in combination with the phosphodiesterase inhibitor 3-isobutyl-1-methyl xanthine, inhibited Na⁺,K⁺-ATPase activity, but this treatment had no effect on specific ouabain binding. No change in ouabain binding was found following activation of protein kinase C by phorbol ester or diacyl glycerol analogue treatment in cells. These data suggest that protein kinase A phosphorylation and inhibition of Na⁺,K⁺-ATPase activity does not lead to any internalization of the enzyme in COS-7 cells.