Effect of high NaCl intake on Na+ and K+ transport in the rabbit distal convoluted tubule

Morphological studies have demonstrated that a chronic increase in distal Na⁺ delivery causes hypertrophy of the distal convoluted tubule (DCT). To examine whether high NaCl-intake also causes functional changes in the well defined DCT, we measured transmural voltage (V _T), lumen-to-bath Na⁺ flux (J _Na(LB)), and net K⁺ secretion (J _K(net)) in DCTs obtained from control rabbits and those on high NaCl-intake diets. The lumen negativeV _T was significantly greater in the high NaCl group than in the control group. The net K⁺ secretion (pmol mm^–1 min^–1) was greater in the high NaCl-intake group (54.1±13.0 vs 14.7±5.6). The K⁺ permeabïlities in both luminal and basolateral DCT membranes, as assessed by the K⁺-induced transepithelial voltage deflection inhibitable with Ba²⁺, were increased in the experimental group. The lumen-to-bath²²Na flux (pmol mm^–1 min^–1) was also greater in the experimental group (726±119 vs 396±65). TheV _T component inhibitable with amiloride was also elevated in the high NaCl-intake group. Furthermore, Na⁺–K⁺-ATPase activity of the DCT was higher in the experimental than in the control group. We conclude that high NaCl intake increases both Na⁺ reabsorption and K⁺ secretion by the DCT. This phenomenon is associated with an increased Na⁺–K⁺-ATPase activity along with increased Na⁺ and K⁺ permeabilities of the luminal membrane, and an increase in the K⁺ permeability of the basolateral membrane. Cellular mechanisms underlying these functional changes remain to be established.     

Evidence for coupled sodium/hydrogen exchange in the rat superficial proximal convoluted tubule

Recent in vitro studies from the rat and rabbit have suggested a tightly coupled sodium/hydrogen ion exchanger on the luminal membrane of proximal tubules. The steep sodium gradient from the lumen to cell supplies indirect energy for hydrogen ions to be pumped from the cell to the lumen. However, a proton translocating pump has been demonstrated in other epithelia, which is independent of sodium transport and directly driven by ATP. To examine the role that sodium might play in the process of acidification, rat proximal convoluted tubules and their surrounding peritubular capillaries were perfused in vivo with artificial ultrafiltrate-like perfusion solutions. Total CO₂ absorption was measured by microcalorimetry during alterations in sodium transport by replacement of the sodium with an impermeant cation, choline, or by inhibition of the (Na⁺+K⁺)-ATPase by removing potassium from both perfusion solutions. Under control conditions the absolute rate of total CO₂ absorption was 140 pmol/mm·min. In the choline substitution and potassium removal experiments, absolute total CO₂ absorption fell to 23 and 28 pmol/mm·min, respectively. The data suggest that: 1) in the rat superficial proximal convoluted tubule approximately 80% of the bicarbonate absorption is tightly coupled to sodium transport; 2) this process is driven indirectly by the (Na⁺+K⁺)-ATPase system; and 3) the residual 20% of acidification appears to be mediated by another mechanism or may be a consequence of technical liminations.     

Video microscopy of intracellular pH in primary cultures of rabbit proximal and early distal tubules

The purpose of this study was to investigate intracytoplasmic pH (pH_i) regulation in primary cultures of proximal (PCT) and distal bright (DCTb) convoluted tubules. PCT and DCTb segments were microdissected from rabbit kidney cortex and cultured in a hormonally defined medium. The cultured epithelia were grown on semi-transparent permeable supports. The pH_i was determined by video microscopy and digital image processing using 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) and measuring the ratio of BCECF fluorescence excited by two successive wavelengths (490 nm and 450 nm). Resting pH_i values, determined in bicarbonatefree medium (extracellular pH: 7.40), were 7.25±0.02 (n=23) and 7.17±0.04 (n=30) for cultured PCT and DCTb respecitively. After the acid-loading procedure, cultured proximal cells recovered their pH_i by means of the classic Na⁺/H⁺ antiporter, sensitive to amiloride and located in the apical membrane only. In cultured DCTb part of the pH_i recovery was mediated by a Na⁺/H⁺ exchange present in the basolateral side. Moreover, at physiological initial pH_i values, chloride removal from the apical solution caused the pH_i to increase in the presence of bicarbonate. In acidified cultured DCTb cells, a partial pH_i recovery was induced in sodium-free media by 15 mM HCO ₃ ^– in the presence of an outward chloride gradient. This pH_i change was completely abolished by 4,4-diisothiocyanostilbene 2,2-disulfonic acid (1 mM). These data suggest that DCTb cells possess in apical anion/base exchanger that resembles the Na⁺-independent Cl^–/HCO ₃ ^– exchanger.     

Thiazide-sensitive Na-Cl cotransport mediates NaCl absorption in amphibian distal tubule

To find out the mechanism(s) underlying NaCl absorption in the distal tubule of Necturus, we devised a variant of the split-drop technique. Following injection an oil column, subsequently split by a NaCl solution isotonic to plasma, a double-barrelled microelectrode (conventional/selective to Na⁺ or to Cl^– ions) recorded Na⁺ ( _Na) or Cl^– ( _Cl) activity and transepithelial potential (V _te). Paired control/low-Na⁺ solutions yielded reabsorptive half-times (t _1/2) of 0.68±0.11 min and 7.6±1.8 min respectively; corresponding V _te values were –22.2±4.0 mV and –7.6±1.9 mV. t _1/2 values of control versus low-Cl^– solutions were 0.77±0.32 min and 6.5±1.7 min respectively, whereas respective V _te values were not different from one another: –23.8±4.3 mV versus –18.8±5.5 mV. Nominally K⁺-free solutions or bumetanide, 10 mol/l, did not alter t _1/2 or V _te, with regard to the paired control. Amiloride, 5 mol/l or 2 mmol/l, failed to decrease t _1/2 or to lower V _te; apparently, the role of a Na⁺/H⁺ antiport does not contribute significantly to NaCl absorption. Furosemide, 0.1 mmol/l, reduced t _1/2 by 54% with regard to the control state. Determination of t _1/2 as a function of increasing hydrochlorothiazide concentrations revealed apical high- and low-affinity sites, estimated at 0.56 mol/l and 0.115 mmol/l respectively. Taken together these observations indicate that NaCl absorption is predominantly carried out by an electroneutral Na⁺-Cl^– cotransport.     

小鼠肾远曲小管三维可视化研究

目的 肾远曲小管（DCT）是肾单位最后一段小管,其与相邻小管的分界及走行的毗邻关系是理解肾形态发生中集合管与肾单位连接方式,以及该段小管参与水盐代谢调节机制的重要结构基础。本实验在肾组织连续切片基础上,采用微细结构三维可视化技术,建立小鼠肾远曲小管的空间走行。 方法 C57/BL/6 J小鼠灌流固定后取肾,垂直于肾长轴切取组织块,树脂812包埋,从肾被膜到肾外髓外带获得2.5 μm厚连续切片720张,获取数字化显微镜图像并通过计算机程序进行配准,追踪来自3只小鼠共90个肾单位远曲小管,观察其空间走行并测量其长度。 结果 肾远曲小管起始于远端小管致密斑后40～180 μm处,上皮由矮柱状或立方陡然变为高柱状,细胞核靠近腔面,走行在皮质迷路其自身的肾小球周围,区域相对独立,末端向被膜方向逐渐移行为连接小管;此处上皮再次变矮,细胞核排列不局限在近腔面。浅层皮质肾单位的远曲小管在回旋返折最高处接触肾被膜1次。远曲小管长度为500～900 μm。 结论 远曲小管较短且盘曲走行,较少与其他小管交错,所以区域较小而独立,可能有助于该节段的吸收功能受激素的精准调节。     

The effect of amiloride on the transepithelial potential difference of the distal tubule of the rat kidney

Summary Microelectrodes with relatively large tips (3–5 O.D.) were used to measure the transepithelial potential difference (PD) of the distal tubule of the rat kidney in the control state and following i.v. administration of amiloride. This drug produced an increase in the magnitude of the positive PD in early distal segments (from +8.0 to +10.5 mV) and a change in polarity of the PD in late distal segments (from –18.0 to +2.5 mV). These data suggest that the potassium-conserving properties of the drug are due to its ability to induce an unfavorable electrochemical gradient opposing passive potassium secretion along the distal tubule.     

A stretch-activated K+ channel in the basolateral membrane ofXenopus kidney proximal tubule cells

The present study examined whether a basolateral potassium ion (K⁺) channel is activated by membrane-stretching in the cell-attached patch. A K⁺ channel of conductance of 27.5 pS was most commonly observed in the basolateral membrane ofXenopus kidney proximal tubule cells. Channel activity increased with hyperpolarizing membrane potentials [at more positive pipette potentials (V _p)]. Open probability (P _o) was 0.03, 0.13, and 0.21 atV _p values of 0, 40, and 80 mV, respectively. Barium (0.1 mM) in the pipette reducedP _o by 79% at aV _p of 40 mV. Application of negative hydraulic pressure (−16 to −32 cm H₂O) to the pipette markedly activated outward currents (fromP _o=0.01 to 0.75) at aV _p of −80 mV, but not inward currents at aV _p of 80 mV. The size of the activated outward currents (from cell to pipette) did not change by replacing chloride with gluconate in the pipette. These results indicate that a stretch-activated K⁺ channel exists in the basolateral membrane of proximal tubule cells. It may play an important role as a K⁺ exit pathway when the cell membrane is stretched (for example, by cell swelling).     

Effect of parathyroid hormone on the connecting tubule from the rabbit kidney: biphasic response of transmural voltage

The effect of parathyroid hormone (PTH) on ion transport was examined by observing transmural (V _T) and basolateral membrane voltage (V _B) in the in vitro perfused rabbit connecting tubule. Addition of 10 nmol/l PTH to the bath induced a biphasic response of V _T, with hyperpolarization followed by depolarization. Chlorophenylthioadenosine cyclic 3,5-monophosphate mimicked the effect of PTH, which did not change the V _B in the connecting tubule cell, but mainly caused changes in the apical membrane voltage. The V _T of distal convoluted tubule and the cortical collecting duct were not affected by PTH. Elimination of Na⁺ from the lumen abolished the PTH-induced V _T responses in the connecting tubule. In the presence of 10 mol/l amiloride, PTH caused an initial hyperpolarization but did not induce the late depolarization. The same was seen in the absence of luminal Ca²⁺. Either addition of 0.1 mmol/l ouabain to the bath or elimination of bath Na⁺ completely abolished the PTH-induced V _T changes. The presence of 5 mmol/l Ba²⁺ in the lumen did not affect the response to PTH. These findings indicate that the initial hyperpolarization may be caused by an increase in Na⁺ influx across the luminal membrane through an amiloride-insensitive Na⁺ conductive pathway and that the late depolarization may be caused by the decrease in Na⁺ influx through the amiloride-sensitive Na⁺ conductive pathway. Luminal Ca²⁺ is necessary for the late depolarization caused by PTH. On the basis of these observations, we suggest that PTH initially increase influxes of both Na⁺ and Ca²⁺ across the luminal membrane and that an increase in intracellular Ca²⁺ in turn suppresses Na⁺ entry through the luminal amiloride-sensitive Na⁺ channel.     

Effect of calcitonin on the regulation of intracellular pH in primary cultures of rabbit early distal tubule

To examine the intracellular pH (pH_i) regulation in primary cultures of rabbit distal convoluted tubules (DCTb) we used the pH-sensitive dye 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF/AM) and a video-microscopy technique. DCTb segments were microdissected from rabbit kidney cortex and cultured in a hormonally defined medium. The culture epithelia were grown on semi-transparent permeable supports. Before pH_i measurement, DCTb primary cultures were maintained for 48–96 h in growth-factor-free medium to obtain quiescent cells. We had previously shown that two mechanisms are involved in the regulation of intracellular pH: a basolateral Na⁺/H⁺ exchanger and an apical Cl^–/HCO ₃ ^– exchanger [1]. The pH_i of DCTb cells was significantly decreased by the addition of 60 nM human calcitonin (from 7.30±0.04 to 7.08±0.04). This response to calcitonin was dose-dependent and mimicked by both forskolin and permeant cyclic AMP derivatives. An initial acidification (of 0.25 pH unit in 7–8 min) was observed after the addition of basolateral amiloride (1 mM). The persistence of the effect induced by human calcitonin in these conditions, suggests that the Na⁺/H⁺ exchanger is not involved in the response. However, the acidification response was blocked in both the absence of chloride at the apical side and by the apical addition of 0.1 mM 4,4-diisothiocyanostilbene-2,2-disulphonic acid (DIDS). These experiments suggest that the target for the human calcitonin effect on pH_i is the Cl^–/HCO ₃ ^– exchanger. This study confirms the importance of this transporter in pH_i regulation within the physiological pH_i range and the influence of calcitonin in the regulation of DCTb cell function.     

Effect of prostaglandin E2 on agonist-stimulated cAMP accumulation in the distal convoluted tubule isolated from the rabbit kidney

The effects of calcitonin, vasoactive intestinal peptide (VIP), parathyroid hormone (PTH) and isoprenaline on intracellular cAMP accumulation were determined in the distal tubule (DCT) microdissected from collagenase-treated rabbit kidney. In DCTb (the initial bright portion) calcitonin (10 ng/ml) elicited a highly reproducible response 203.7±19.1 fmol cAMP mm^–1 4 min^–1 (SE, N=13) whereas VIP-induced cAMP accumulation was less and more variable from one experiment to another (1 M, 97.2±17.8 fmol mm^–1 4 min^–1, SE, N=12). When used in combination, these two agonists were non-additive, indicating stimulation of a single pool of cAMP in DCTb. In DCTg, (granular) which consists of at least two cell types, PTH (100 nM) elicited a marked, reproducible accumulation of cAMP (154.3±27.0 fmol mm^–1 4 min^–1; SE, N=5). Isoprenaline (1 M) and VIP (1 M) induced much smaller increases in cAMP levels 20.9±2.7 and 29.4±4.1 fmol mm^–1 4 min^–1 (SE, N=5) respectively, and, when used in combination, were non-additive, demonstrating that VIP and isoprenaline are active on the same cell type. In DCTb, prostaglandin E₂ (PGE₂) inhibited both calcitoninand VIP-stimulated cAMP accumulation (calcitonin 57.8±2.7% inhibition, SE, N=16; VIP, 80.6±2.1% inhibition, SE, N=5). The EC₅₀ values for calcitonin were 1.21±0.33 ng/ml and 1.83±0.25 ng/ ml (SD, N=3) in the absence and presence of PGE₂ (300 nM) respectively with an IC₅₀ for PGE₂ of 26.3±6.3 nM (SE, N=4). In contrast, no effects of PGE₂ were seen in DCTg vis à vis PTH, isoprenaline or VIP. The percentage inhibition of calcitonin-stimulated cAMP accumulation by PGE₂ was of the same order in the presence of isobutylmethylxanthine (an inhibitor of all types of phosphodiesterase), Ro 20-1724 (inhibitor of low-K _m cAMP-specific phosphodiesterase) or in the absence of inhibitor. Preincubation of DCTb with pertussis toxin for up to 8 h in different experimental conditions did not relieve the inhibition by PGE₂. Protein kinase C activation by phorbol ester did not attenuate calcitonin responses. These data demonstrate that the inhibition by PGE₂ of cAMP production is restricted to the initial portion (DCTb) of the distal convoluted tubule and is effective on both calcitonin and VIP responses. When tested in the presence of Ro 20-1724, ionomycin, A₁-adenosine, ₂-adrenergic and muscarinic agonists were without effect on calcitoninand PTH-stimulated cAMP accumulation in DCTb and DCTg respectively.     

The thyroid cell monolayer in culture

When cultured on collagen coated nitrocellulose filters, thyroid epithelial cells form morphologically and functionally polarized monolayers. The bioelectric parameters of these monolayers were measured after mounting in Ussing chambers; transepithelial potential (V _ab), short circuit current (I _sc) and transepithelial resistance were respectively 12±1 mV (apical side negative), 3.8±0.2 A cm^–2 and 3250±214 cm² (mean±SEM,n=75). Eighty two percent of the short circuit current was related to sodium absorption as shown by inhibition by apical amiloride (K _m=0.2 M) and by basal ouabain (K _1/2=0.3 M). Amphotericin B (5–25 g/ml) added to the apical bath increasedI _sc suggesting an apical rate-limiting step. Step by step replacement of choline by Na⁺ in a Na⁺-free medium resulted in a progressive increase inV _ab andI _sc with half maximal effect at 20±1 mM Na⁺. Thyrotropin (TSH) increasedI _sc andV _ab in a biphasic way with a transient maximum after 5 min and a plateau after 20 min (about four times the basal level at 100 U/ml TSH). This increase in sodium transport was also inhibited by apical amiloride. Thus, in culture, the thyroid cell monolayer behaves as a tight sodium absorbing epithelium controlled by TSH, with a rate limiting apical sodium channel as the entry mechanism and a basolateral Na⁺, K⁺-ATPase as the electromotive force.     

Electrophysiological analysis of rat renal sugar and amino acid transport

Transepithelial and cellular electrical potential changes were measured in response to luminal perfusion ofd-glucose and related substrates in micropuncture experiments on rat kidney in vivo. By studying the dependence of the potential response on various experimental parameters, some insight was obtained into the mechanism of Na⁺ coupled glucose absorption. The experiments confirm the driving forces for glucose absorption in the living cell to be: a) the Na concentration gradient, b) the electrical potential gradient and c) the glucose concentration gradient across the brushborder membrane. Furthermore they describe the substrate specificity of the cotransport mechanism and the mechanism of inhibition ofd-glucose transport by various inhibitors, such as phlorizin, harmaline and oubain. The latter experiments suggest that the active Na⁺ pump in the peritubular cell membrane, which establishes the Na⁺ ion gradient and the electrical potential gradient across the brushborder, contributes a measurable partial conductance to the overall electrical conductance of the peritubular cell membrane.     

Glucagon inhibits water and NaCl transports in the proximal convoluted tubule of the rat kidney

The effects of glucagon on water and electrolyte transport in the kidney were investigated on hormone-deprived rats, i.e. thyroparathyroidectomized diabetes insipidus Brattleboro rats infused with somatostatin. Glucagon consistently inhibited the reabsorption of water and Na⁺, Cl^–, K⁺ and Ca²⁺ along the proximal tubule accessible to micropuncture, leaving the reabsorption of inorganic phosphate (P_i) untouched. In the loop, besides its previously described stimulatory effects on Na⁺, Cl^–, K⁺, Ca²⁺ and Mg²⁺ reabsorption, glucagon strongly inhibited P_i reabsorption, very probably in the proximal straight tubule. These effects resulted in a significant phosphaturia and considerable reductions of Mg²⁺ and Ca²⁺ excretions. The effects of glucagon at both the whole kidney and the nephron levels are very similar to those previously described for calcitonin. In the absence of an adenylate cyclase system sensitive to glucagon and calcitonin in the rat proximal tubule, and from the analogy of their physiological effects with those elicited by parathyroid hormone, it is suggested that glucagon and calcitonin exert their inhibitory effects on Na and P_i reabsorption in the proximal tubule through another pathway, which could be the phosphoinositide regulatory cascade.     

Mechanisms of oxalate absorption and secretion across the rabbit distal colon

To further evaluate the mechanisms of oxalate (Ox^2–) transport in the intestine the following studies were performed using isolated, short-circuited segments of the rabbit distal colon (DC). In control buffer, the DC absorbed Ox^2– (net Ox^2– flux, J _Net ^Ox =5.4±0.7 pmol · cm^–2 · h^–1). Replacement of Na⁺ with N-methyl-d-glucamine (NMDG⁺) abolished Ox^2– absorption by decreasing mucosal to serosal Ox^2– flux (J _ms ^Ox ), without affecting Cl^– transport, while gluconate substitution for Cl^– did not affect J _Net ^Ox or net Na⁺ flux (J _Net ^Na ). Addition of Na⁺ to the serosal side of tissues bathed by NMDG⁺ buffer increased J _ms ^Ox 40% without altering mucosal to serosal Cl^– flux (J _ms ^Cl ). Serosal amiloride or dimethyl amiloride (10^–3 M) abolished J _Net ^Ox by decreasing J _ms ^Ox , it increased serosal to muscosal Cl^– flux (J _sm ^Cl ) and it gradually inhibited short-circuit current (I _sc). Mucosal amiloride (10^–4 M) abolished I _sc but had no effect on Ox^2– or Cl^– fluxes. Serosal 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS, 10^–6 M) reduced J _ms ^Ox by 20% and J _Net ^Ox by 43% without affecting J _ms ^Cl or J _Net ^Cl . Dibutyryl cyclic adenosine monophosphate (dB-cAMP, 5×10^–4 M, both sides) stimulated Ox^2– secretion (J _Net ^Ox = –12.6±3.3 pmol · cm^–2 · h^–1). The dB-cAMP-induced secretion of Ox^2– and Cl^– were fully abolished by serosal furosemide (10^–4 M) and partially inhibited (35%) by 5×10^–4 M mucosal NPPB [5-nitro-2-(3-phenylpropylamino)-benzoic acid], a putative Cl^– channel blocker. It is proposed that: (1) basal absorption of Ox^2–, but not Cl^–, is dependent upon a previously undescribed basolateral Na⁺-H⁺ exchanger that may be coupled to a DIDS-sensitive, basolateral anion exchange system that mediates Ox^2– flux; (2) the DC secretes Ox^2– in response to dB-cAMP by a mechanism that is indistinguishable from the pathway for Cl^– secretion.     

The effect of shear stress on the basolateral membrane potential of proximal convoluted tubule of the rat kidney

As consequence of glomerular filtration the viscosity of blood flowing through the efferent arteriole increases. Recently, we found that shear stress modulates proximal bicarbonate reabsorption and nitric oxide (NO·) was the chemical mediator of this effect. In the present work, we found that agonists of NO· production affected basolateral membrane potential (V _blm) of the proximal convoluted tubule (PCT) epithelium. Using paired micropuncture experiments, we perfused peritubular capillaries with solutions with different viscosity while registering the V _blm. Our results showed that a 50% increment in the viscosity, or the addition of bradykinin (10⁻⁵ M) to the peritubular perfusion solution, induced a significant and similar hyperpolarization of the V _blm at the PCT epithelium of 6 ± 0.7 mV (p < 0.05). Both hyperpolarizations were reverted by l-NAME (10⁻⁴ M). Addition of 2,2′-(hydroxynitrosohydrazino) bis-ethanamine (NOC-18) 3 × 10⁻⁴ M to the peritubular perfusion solution induced a hyperpolarization of the same magnitude of that high viscosity or bradykinin. These results strongly suggest the involvement of NO· in the effect of high viscosity solutions. This effect seems to be mediated by activation of channels as glybenclamide (5 × 10⁻⁵ M) added to peritubular solutions induced a larger depolarization of the V _blm with high viscosity solutions. Acetazolamide (5 × 10⁻⁵ M) added to high viscosity solutions induced a larger hyperpolarization (8 ± 1 mV; p < 0.05), suggesting that depolarizing current due to exit across the basolateral membrane damps the hyperpolarizing effect of high viscosity. Considering that Na⁺ and consequently water reabsorption is highly dependent on electrical gradient, the present data suggest that the endothelium of kidney vascular bed interacts in paracrine fashion with the epithelia, affecting V _blm and thus modulating PCT reabsorption.     

Electrophysiological study of transport systems in isolated perfused pancreatic ducts: properties of the basolateral membrane

In order to study the mechanism of pancreatic HCO ₃ ^– transport, a perfused preparation of isolated intra-and interlobular ducts (i.d. 20–40 m) of rat pancreas was developed. Responses of the epithelium to changes in the bath ionic concentration and to addition of transport inhibitors was monitored by electrophysiological techniques. In this report some properties of the basolateral membrane of pancreatic duct cells are described. The transepithelial potential difference (PD_te) in ducts bathed in HCO ₃ ^– -free and HCO ₃ ^– -containing solution was –0.8 and –2.6 mV, respectively. The equivalent short circuit current (I_sc) under similar conditions was 26 and 50 A·cm^–2. The specific transepithelial resistance (R_te) was 88 cm². In control solutions the PD across the basolateral membrane (PD_bl) was –63±1 mV (n=314). Ouabain (3 mmol/l) depolarized PD_bl by 4.8±1.1 mV (n=6) within less than 10 s. When the bath K⁺ concentration was increased from 5 to 20 mmol/l, PD_bl depolarized by 15.9±0.9 mV (n=50). The same K⁺ concentration step had no effect on PD_bl if the ducts were exposed to Ba²⁺, a K⁺ channel blocker. Application of Ba²⁺ (1 mmol/l) alone depolarized PD_bl by 26.4±1.4 mV (n=19), while another K⁺ channel blocker TEA⁺ (50 mmol/l) depolarized PD_bl only by 7.7±2.0 mV (n=9). Addition of amiloride (1 mmol/l) to the bath caused 3–4 mV depolarization of PD_bl. Furosemide (0.1 mmol/l) and SITS (0.1 mmol/l) had no effect on PD_bl. An increase in the bath HCO ₃ ^– concentration from 0 to 25 mmol/l produced fast and sustained depolarization of PD_bl by 8.5±1.0 mV (n=149). It was investigated whether the effect of HCO ₃ ^– was due to a Na⁺⁺-dependent transport mechanism on the basolateral membrane, where the ion complex transferred into the cell would be positively charged, or whether it was due to decreased K⁺ conductance caused by lowered intracellular pH. Experiments showed that the HCO ₃ ^– effect was present even when the bath Na⁺ concentration was reduced to a nominal value of 0 mmol/l. Similarly, the HCO ₃ ^– effect remained unchanged after Ba²⁺ (5 mmol/l) was added to the bath. The results indicate that on the basolateral membrane of duct cells there is a ouabain sensitive (Na⁺+K⁺)-ATPase, a Ba²⁺ sensitive K⁺ conductance and an amiloride sensitive Na⁺/H⁺ antiport. The HCO ₃ ^– effect on PD_bl is most likely due to rheogenic anion exit across the luminal membrane.     

The ion conductances of colonic crypts from dexamethasone-treated rats

Whole-cell patch-clamp studies were performed in isolated colonic crypts of rats pretreated with dexamethasone (6 mg/kg subcutaneously on 3 days consecutively prior to the experiment). The cells were divided into three categories according to their position along the crypt axis: surface cells (s.c.); mid-crypt cells (m.c.) and crypt base cells (b.c.). The zero-current membrane voltage (V _m) was –56 ± 2 mV in s.c (n = 34); –76 ± 2 mV in M.C. (n = 47); and –87 ± 1 mV in b.c. (n = 87). The whole-cell conductance (G _m) was similar (8–12 nS) in all three types of cells. A fractional K⁺ conductance accounting for 29–67% ofG _m was present in all cell types. A Na⁺conductance was demonstrable in s.c. by the hyperpolarizing effect onV _m of a low-Na⁺ (5 mmol/1) solution. In m.c. and b.c. the hyperpolarizing effect was much smaller, albeit significant. Amiloride had a concentration-dependent hyperpolarizing effect onV _m in m.c. and even more so in s.c.. It reducedG _m by approximately 12%. The dissociation constant (K _D) was around 0.2 mol/l. Triamterene had a comparable but not additive effect (K _D = 30 mol/l,n = 14). Forskolin (10 mol/l, in order to enhance cytosolic adenosine 3, 5-cyclic monophosphate or CAMP) depolarizedV _m in all three types of cells. The strongest effect was seen in b. c..G _m was enhanced significantly in b.c. by 83% (forskolin) to 121% [8-(4-chlorophenylthio)cAMP]. The depolarization ofV _m and increase inG _m was caused to large extent by an increase in Cl^– conductance as shown by the effect of a reduction in bath Cl^– concentration from 145 to 32 mmol/1. This manocuvre hyperpolarizedV _m under control conditions significantly by 6–9 mV in all three types of cells, whilst it depolarizedV _m in the presence of forskolin in m.c. and in b.c.. These data indicate that s.c. of dexamethasone-treated rats possess mostly a K⁺ conductance and an amiloride- and Tramterene-inhibitable Na⁺ conductance. m.c. and b.c. possess little or no Na⁺ conductance; theirV _m is largely determined by a K⁺ conductance. Forskolin (via cAMP) augments the Cl^– conductance of m.c. and b.c. but has only a slight effect on s.c.     

Polarized targeting of ion channels in neurons

Since the time of Cajal it has been understood that axons and dendrites perform distinct electrophysiological functions that require unique sets of proteins [Cajal SR Histology of the nervous system, Oxford University Press, New York, (1995)]. To establish and maintain functional polarity, neurons localize many proteins specifically to either the axonal or the somatodendritic compartment. In particular, ion channels, which are the major regulators of electrical activity in neurons, are often distributed in a polarized fashion. Recently, the ability to introduce tagged proteins into neurons in culture has allowed the molecular mechanisms underlying axon- and dendrite-specific targeting of ion channels to be explored. These investigations have identified peptide signals from voltage-gated Na⁺ and K⁺ channels that direct trafficking to either axonal or dendritic compartments. In this article we will discuss the molecular mechanisms underlying polarized targeting of voltage-gated ion channels from the Kv4, Kv1, and Na_v1 families.     

Effect of parathyroid hormone on acid/base transport in rabbit renal proximal tubule S3 segment

The effect of parathyroid hormone (PTH) on acid/base transport in isolated rabbit renal proximal tubule S3 segment was investigated with double-barreled and conventional microelectrodes. PTH (10 nM) induced a small depolarization and enhanced the initial rates of cell pH (pH_i) increase and cell Cl^– ([Cl^–]_i) decrease in response to bath Cl^– removal by 28.0±2.1% and 31.0±6.4% respectively. The calculated initial HCO₃ ^– influx to bath Cl^– removal was also enhanced by 28%. On the other hand, PTH reduced the initial rate of pH_i decrease to luminal Na⁺ removal in the absence of HCO₃ ^–/CO₂ by 20.4±3.9%. The PTH-induced depolarization was not accompanied with changes in steadystate pH_i or [Cl^–]_i levels, but was greatly attenuated in the presence of ouabain (0.1 mM). Either dibutyrylcAMP (0.1 mM) plus theophylline (1 mM) or forskolin (10 M) alone could reproduce all the effects of PTH. These results indicate that (a) PTH inhibits the luminal Na⁺/H⁺ exchanger but stimulates the basolateral Cl^–/HCO₃ ^– exchanger in the S3 segment; (b) the PTH-induced depolarization largely results from inhibition of Na⁺/K⁺-ATPase and (c) all these effects are at least partly mediated by a cAMP-dependent mechanism.     

Influence of Mitragyna Ciliata (MYTA) on the Microsomal Activity of ATPase Na+/K+ Dependent Extract on a Rabbit Heart

Mitragyna ciliata (MYTA) (Rubiaceae) inhibits plasmodia activity. MYTA induces a cardiotonicity of the digitalic type on rat''s isolated heart. In this work we studied the effect of MYTA on microsomal Na⁺/K⁺ dependant ATPase (Na⁺, K⁺ ATPase) extracted from the heart of a rabbit since digitalics inhibit Na⁺, K⁺ ATPase. Our results revealed that the Na⁺/K⁺ ATPase has an optimum pH of 7.4 and temperature of 37°C respectively. There is a linear relationship between the organic phosphate formed and the incubation time over 25 mins incubation period. The ATP hydrolysis rate in the presence of MYTA was 0.775 µM/min. LINEWEAVER and BURK plots showed that MYTA did not alter K_M (1.31 mM) but decreased V_MAX. This study shows that MYTA exerts a non-competitive inhibition on the microsomal Na⁺/K⁺ ATPase extracted from rabbit heart with a Ci₅₀ of 48 µg / ml. We conclude that the mechanism of action of MYTA is linked to the inhibition of the Na⁺/K⁺ ATPase like cardiotonics of the digitalic type.