Sodium-dependent adenosine transport is diminished in brush border membrane vesicles from hypothyroid rat kidney

 Studies of the uptake of [3H]adenosine ([³H]ADO) were performed using brush border membrane vesicles (BBMV) from normal (N) and hypothyroid (Tx) rat kidneys, to test if decreased Na⁺ reabsorption in hypothyroidism might be associated with abnormalities in ADO transport. [³H]ADO uptake (1–10 μmol) for both conditions was measured in the presence of Na⁺ (10–150 mmol/l); the effects of dipyridamole (10 μmol/l) and 1,3-dipropyl-8-(2-amino-4-chlorophenyl)xanthine (PACPX, 10 μmol/l) were also studied. Na⁺-stimulated ADO uptake was decreased in Tx BBMV. Michaelis–Menten constants showed a decreased ADO carrier affinity (K _m 2.46 ± 0.14 in N, vs K _m 4.46 ± 0.88 μmol/l in Tx, P<0.05), with no change in the number of carriers (V _max 295 ± 25 in N, vs 229.2 ± 56 pmol·min^–1·mg protein in Tx). Na⁺ affinity remained unchanged (K _Na+ 11.5 ± 3.5 in N, vs K _Na+ 12.72 ± 0.7 mmol/l in Tx). Inhibition of Na⁺-dependent ADO transport was 50% in N as opposed to 58% in Tx with dipyridamole, and 72% in N versus 47% in Tx with PACPX. These results suggest that decreased Na⁺-dependent ADO cotransport contributes to the diminished tubular reabsorption that occurs in hypothyroidism. Received: 17 June 1996 / Received after revision: 9 September 1996 / Accepted: 16 September 1996     

Electrophysiological studies in principal cells of rat cortical collecting tubules ADH increases the apical membrane Na+-conductance

The mechanism of ion transport across principal cells of rat cortical collecting tubules (CCT) and its regulation by vasopressin (ADH) has been studied in the isolated perfused tubule. To amplify the response to ADH rats were treated with 5 mg I. M. desoxycorticosterone 4–9 days prior to the experiments. Addition of 2·10^–10 mol·1^–1 ADH increased the transepithelial voltage from –5.1 ±0.7 mV to –16.1±1.4 mV (n=37) and decreased the transepithelial resistance from 51±4 cm² to 39±2 cm² (n=33). Optical and functional differentiation of impalements of principal and intercalated cells was made and only data of principal cells are presented. ADH depolarized the apical membrane from 79±1 mV to 66±2 mV (n=26) and decreased the fractional resistance of the apical membrane from 0.76±0.04 to 0.70±0.04 (n=13). These ADH effects were prevented by 10^–5 or 10^–4 mol·1^–1 luminal amiloride which hyperpolarized the apical membrane when added in the presence or absence of ADH. Apical and basolateral membranes were dominated by large K⁺ conductances and addition of 3 mmol·1^–1 barium to bath or lumen perfusates increased transepithelial resistance almost two-fold, whereas luminal amiloride increased the transepithelial resistance only by 26–35%. Ouabain (0.5 mmol·1^–1, bath) depolarized the basolateral membrane and decreased its K⁺ conductance. These effects were prevented by the simultaneous presence of apical amiloride suggesting that the only route of Na⁺ entry into the principal cells occurred via the amiloride sensitive Na⁺ conductance. We conclude that ADH stimulates Na⁺ reabsorption and K⁺ secretion in the rat CCT primarily by increasing the Na⁺ conductance in the apical cell membrane.Parts of this study have been presented at the 19th ASN meeting in Washington, DC, USA 1986     

Regulatory volume decrease in a renal distal tubular cell line (A6) II. Effect of Na+ transport rate

A6 epithelia, a cell line originating from the distal tubular part of the kidney ofXenopus laevis, were cultured on permeable supports and mounted in an Ussing-type chamber. Cell thickness (T _c), short-circuit current (I _sc) and transepithelial conductance (G _t) were recorded while tissues were bilaterally incubated in NaCl solutions and the transepithelial potential was clamped to zero. Effects of inhibition and stimulation of transepithelial Na⁺ transport on cell volume and on its regulation during a hyposmotic challenge were investigated. Under control conditions a slow spontaneous decrease ofT _c described by a linear baseline was recorded. The reduction of the apical osmolality from 260 to 140 mosmol/kg did not alter cell volume significantly, demonstrating a negligible water permeability of the apical barrier. The inhibition of Na⁺ uptake by replacing apical Na⁺ byN-methyl-d-glucamine (NMDG⁺) did not affect cell volume under isotonic conditions. An increase ofT _c by 12.1% above the control baseline was recorded after blocking active transport with ouabain for 60 min. The activation of Na⁺ transport with insulin or oxytocin, which is known to activate the apical water permeability in other epithelia, did not alter cell volume significantly. The insensitivity of cell volume to alterations in apical Na⁺ uptake or Na⁺ pump rate confirms the close coupling between apical and basolateral transport processes. The blockage of basolateral K⁺ channels by 5 mM Ba²⁺ elicited a significant increase inT _c of 16.3% above control. Quinine, a potent blocker of volume-activated K⁺ channels, did not changeT _c significantly. Basolateral hypotonicity elicited a rapid rise inT _c followed by a regulatory volume decrease (RVD). An RVD was also recorded after blocking apical Na⁺ uptake as well as after stimulating apical Na⁺ uptake with oxytocin or insulin. Inhibition of active transport with ouabain as well as blocking K⁺ efflux at the basolateral side with Ba²⁺ or quinine abolished the RVD. The inhibition of the RVD by ouabain seems to be caused by a depletion of cellular K⁺, whereas the effects of Ba²⁺ and quinine are most likely due to the blockage of the basolateral K⁺ pathway.     

Contractile effects of adenosine, coronary flow and perfusion pressure in murine myocardium

There is mixed evidence adenosine receptors (ARs) may enhance myocardial contractility, although this remains contentious. We assessed inotropic actions of adenosine (50 μM) and selective AR activation with 100 nM N ⁶-cyclohexyladenosine (CHA; A₁AR agonist), 25 nM 2-[p-(2-carboxyethyl) phenethylamino]-5′-N-ethylcarboxamidoadenosine (CGS-21680; A_2AAR agonist) and 100 nM 2-chloro-N ⁶-(3-iodobenzyl)-adenosine-5′-N-methyluronamide (Cl-IB-MECA; A₃AR agonist) in mouse hearts perfused at constant pressure, constant flow, or conditions of stable flow and pressure (following maximal nitroprusside-mediated dilatation at constant flow). Adenosine and CGS-21680 significantly (although modestly) increased force in constant-pressure perfused hearts (≤10 mmHg elevations in systolic pressure), effects paralleled by coronary vasodilatation (≤10 ml min⁻¹ g⁻¹ elevations in flow). Neither CHA nor Cl-IB-MECA altered force or flow. With constant-flow perfusion, adenosine and CGS-21680 reduced systolic pressure in parallel with perfusion pressure. When changes in coronary flow and pressure were prevented, CGS-21680 failed to alter contractility. However, adenosine still enhanced systolic pressure up to 10 mmHg. Relations between flow, perfusion pressure and ventricular force evidence substantial Gregg effects in murine myocardium: systolic force increases transiently by approximately 1 mmHg ml⁻¹ min⁻¹ g⁻¹ rise in flow during the first minutes of hyperaemia and in a sustained manner (by ∼1 mmHg mmHg⁻¹) during altered perfusion pressure. These effects contribute to inotropism with AR agonism when flow/pressure is uncontrolled. In summary, we find no evidence of direct A₁ or A₃AR-mediated inotropic responses in intact myocardium. Inotropic actions of A_2AAR agonism appear entirely Gregg-related. Nonetheless, the endogenous agonist adenosine exerts a modest inotropic action independently of flow and perfusion pressure. The basis of this response remains to be identified.     

The effect of brefeldin A on terbutaline-induced sodium absorption in fetal rat distal lung epithelium

 We studied the effect of brefeldin A, which inhibits the intracellular trafficking of membrane proteins from the cytosolic pool to the cell surface, on terbutaline (a β₂-specific adrenergic agonist)-induced alterations in ion transport by primary monolayer cultures of fetal rat distal lung epithelium. The amiloride-sensitive short circuit current (Isc) increased 2.5-fold 50 min after application of terbutaline (10 μM) from basolateral side; this response was abolished by pretreatment with brefeldin A (1 μg/ml). Brefeldin A did not suppress the Na⁺/K⁺ pump capacity. Single channel patch clamp experiments demonstrated that terbutaline increased the density of amiloride-sensitive Na⁺-permeable nonselective cation channels on the apical cell membrane and this action was blocked by brefeldin A. These observations suggest that β2-specific adrenergic agonists promote the trafficking of amiloride sensitive Na⁺-permeable nonselective cation channels to the apical cell surface. Received: 4 February 1997 / Received after revision: 16 April 1997 / Accepted: 16 May 1997     

The role of mitochondria-rich cells in sodium transport across amphibian skin

 The possible participation of mitochondria-rich cells in transepithelial Na⁺ transport across frog skin under ”physiological conditions” (low apical [Na⁺], open circuited) was analysed by recording electrophysiological parameters from principal cells with intracellular microelectrodes and using measurement of Rb⁺ uptake into the epithelial cells from the serosal side via the Na⁺/K⁺-ATPase. It was observed that transport perturbation with amiloride induced changes in the apical potential difference and fractional apical resistance in principal cells, observations which are compatible with the notion that the essential fraction of transcellular current flow occurs across these cells. Amiloride-inhibitable uptake of Rb⁺ was also restricted to principal cells, the amount being about equivalent to the predicted rate of K⁺ recycling via the Na⁺/K⁺-ATPase. The results indicate that principal cells are responsible for transepithelial Na⁺ transport irrespective of the experimental conditions. Flow of Na⁺ across mitochondria-rich cells appears to be negligible. Received: 29 February 1996 / Received after revision: 23 June 1996 / Accepted 9 September 1996     

A polarized salivary cell monolayer useful for studying transepithelial fluid movement in vitro

 There are no reported, convenient in vitro models for studying polarized functions in salivary epithelial cells. Accordingly, we examined three often-used salivary cell lines for their ability to form a polarized monolayer on permeable, collagen-coated polycarbonate filters. Only the SMIE line, derived from rat submandibular gland, had this ability. The SMIE cell monolayer exhibited junctional complexes, with a tight-junction-associated protein, ZO-1, localized to cell–cell contact areas. The Na⁺/K⁺-ATPase α₁-subunit was detected predominantly in the basolateral membranes, while the Na⁺/H⁺ exchanger isoform 2 appeared primarily in the apical membranes. Using adenovirus-mediated cDNA transfer, SMIE cells were shown to be capable of routing marker proteins (β-galactosidase ± a nuclear targeting signal, α1-antitrypsin, aquaporin-1) to appropriate locations. Furthermore, this salivary cell monolayer provided a convenient tool for studying aquaporin-1-mediated, osmotically directed, transepithelial fluid movement in vitro. Thus, SMIE cells appear to be a useful experimental model with which to study some polarized functions in a salivary epithelial cell line. Received: 16 September 1997 / Accepted: 17 September 1997     

Distal colonic Na+ absorption inhibited by luminal P2Y2 receptors

Luminal P2 receptors are ubiquitously expressed in transporting epithelia. In steroid-sensitive epithelia (e.g., lung, distal nephron) epithelial Na⁺ channel (ENaC)-mediated Na⁺ absorption is inhibited via luminal P2 receptors. In distal mouse colon, we have identified that both, a luminal P2Y₂ and a luminal P2Y₄ receptor, stimulate K⁺ secretion. In this study, we investigate the effect of luminal adenosine triphosphate/uridine triphosphate (ATP/UTP) on electrogenic Na⁺ absorption in distal colonic mucosa of mice treated on a low Na⁺ diet for more than 2 weeks. Transepithelial electrical parameters were recorded in an Ussing chamber. Baseline parameters: transepithelial voltage (V _te): −13.7 ± 1.9 mV (lumen negative), transepithelial resistance (R _te): 24.1 ± 1.8 Ω cm², equivalent short circuit current (I _sc): −563.9 ± 63.8 μA/cm² (n = 21). Amiloride completely inhibited I _sc to −0.5 ± 8.5 μA/cm². Luminal ATP induced a slowly on-setting and persistent inhibition of the amiloride-sensitive I _sc by 160.7 ± 29.7 μA/cm² (n = 12, NMRI mice). Luminal ATP and UTP were almost equipotent with IC₅₀ values of 10 μM and 3 μM respectively. In P2Y₂ knock-out (KO) mice, the effect of luminal UTP on amiloride-sensitve Na⁺ absorption was absent. In contrast, in P2Y₄ KO mice the inhibitory effect of luminal UTP on Na⁺ absorption remained present. Semiquantitative polymerase chain reaction did not indicate regulation of the P2Y receptors under low Na⁺ diet, but it revealed a pronounced axial expression of both receptors with highest abundance in surface epithelia. Thus, luminal P2Y₂ and P2Y₄ receptors and ENaC channels co-localize in surface epithelium. Intriguingly, only the stimulation of the P2Y₂ receptor mediates inhibition of electrogenic Na⁺ absorption.     

cAMP increases the basolateral Cl−-conductance in the isolated perfused medullary thick ascending limb of Henle's loop of the mouse

The effect of cAMP on transepithelial and transmembrane potential differences and resistances was examined in isolated in vitro perfused mouse medullary thick ascending limbs of Henle's loop (mTAL). The effects of furosemide and barium were tested. Stimulation of NaCl transport by ADH 10^–9+dbcAMP 4·10^–4+forskolin 10^–6 mol·l^–1 (paired experiments) resulted in: a) an increase in transepithelial potential difference, referenced to the grounded bath, from +6.7±0.3 mV to +12.0±0.4 mV (n=47); b) a decrease in transepithelial resistance from 25±1 cm² to 20±1 cm² (n=47); c) a depolarization of the basolateral membrane by 12 mV and of the apical membrane by 7 mV (n=36); d) a decrease in the fractional resistance of the basolateral membrane from 0.27±0.005 to 0.15±0.06 (n=12). Furosemide (10^–4 mol·l^–1) abolished the active transepithelial transport potential and hyperpolarized the basolateral membrane potential to values which were similar in both control and cAMP treated mTAL segments. Barium increased the transepithelial resistance and depolarizedPD _bl to similar values in both functional states. An increase in the fractional conductance of the basolateral membrane was also seen, if, prior to the cAMP treatment, the luminal Na⁺2Cl^–K⁺ contransport was inhibited by furosemide. Thus, we propose that stimulation of active NaCl reabsorption in the mTAL segment of the mouse by ADH, mediated via cAMP, increases primarily the basolateral chloride conductance.Supported by Deutsche Forschungsgemeinschaft Gr 480/6-2Parts of this study have been presented at the 59th Meeting of the German Physiological Society in Dortmund 1984 and at the 69th FASEB Meeting in Anaheim 1985     

Sodium gradient-energized concentrative transport of adenosine in renal brush border vesicles

The uptake of adenosine in brush border vesicles of the proximal tubule of the rat kidney has been studied with a filtration technique. The initial rate of uptake was almost 6 times greater in the presence of NaCl than in the presence of KCl. The stimulatory effect of Na⁺ was strictly dependent on a gradient of Na⁺ (out>in). The time course of uptake showed an overshoot with a maximum at 20 s with a gradient of NaCl, but not with KCl. Inosine and 5-AMP were produced from adenosine within the vesicles. In the presence of an inhibitor or adenosine deaminase adenosine was not significantly metabolized during the first 20 s of uptake. Thus, kinetic parameters of transport could be studied in the absence of interferences with metabolism. AK _m of 1.1 M and aV _max of 232 pmol · min^–1 · mg protein^–1 were calculated for the Na⁺ gradient-dependent transport. The dependency on a Na⁺ gradient, the capacity for uphill transport and the high affinity for adenosine situate this transport system apart from the mechanisms of transport of nucleosides described so far. It may be relevant in regard to the role of adenosine in the regulation of glomerular filtration.Abbreviations used EHNA erythro-9-(2-hydroxy-3-nonyl)adenine - FCCP carbonylcyanide p-trifluoromethoxy-phenylhydrazone - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - Tris tris (hydroxymethyl)-aminomethane     

Bradykinin stimulation of electrogenic ion transport in epithelial layers of cultured human endometrium

Primary cultures of glandular endometrial epithelial cells grown on permeable supports formed monolayers with a high transepithelial electrical resistance [1096±83 .cm² (n=34)] and displayed electrogenic ion transport as demonstrated by an inward short circuit current (I_sc; 20±2A/cm²). Bradykinin, 10^–8–10^–6M, added to either the basolateral or apical solutions enhanced the inward I_sc. The concentration-response curves for bradykinin were bell-shaped in nature. The I_sc response was more sensitive to apical addition of bradykinin and the maximum response was also greater with apical bradykinin. The increases in I_sc were accompanied by two- to three-fold increases in transepithelial conductance. Apical addition of amiloride, 10^–4M, reduced the unstimulated I_sc by 80%. In the presence of amiloride, the response to both apical and basolateral bradykinin was reduced by >50 % in 8 out of 18 layers, and the mean response was reduced by approximately 25%. The data are consistent with a physiological role for bradykinin in the control of the intra-uterine electrolyte environment, mediated in part by enhanced Na⁺ absorption.     

Effects of standard diuretics and RPH 2823 on transepithelial Na+ transport in isolated frog skin

Summary Short-circuit current (SCC) techniques were used to monitor the effects of various diuretic agents on Na⁺ transport in isolated frog skin, a model for the late distal tubule and the collecting duct of the mammalian kidney. Acctazolamide, hydrochlorothiazide, torasemide, and ethacrynic acid did not affect sodium transport (as indicated by the SCC) or transepithelial electrical resistance when added either to the apical (outer) or to the inner (basolateral, corial) bathing solution of the tissue. However, Na⁺ transport was sensitive to amiloride, the triamterene derivate dimethylamino-hydroxypropoxytriamterene (RPH 2823), and to furosemide. Whereas apical amiloride, and RPH 2823 induced a dose-dependent decrease in SCC and increase in transepithelial electrical resistance, apical furosemide resulted in a dose-dependent increase in SCC and a decrease in electrical resistance. None of the three diuretic agents caused a significant change in SCC when applied to the inner bathing Ringer's solution. The small furosemide-induced decrease in resistance compared with the huge increase in SCC suggests that furosemide affects Cl^– permeability as well as Na⁺ permeability. Evidence for this notion was achieved by the following findings: (a) The decrease in resistance after furosemide was more pronounced in tissues bathed in Cl^–-free solutions compared with Cl^–-containing solutions. (b) In contrast, SCC stimulation by apical furosemide is Cl^–-ion independent, but strongly Na⁺-ion dependent. (c) SCC stimulation by furosemide is amiloride-sensitive. With respect to the onset, locus, and reversibility of action, it seems reasonable to assume that amiloride, RPH 2823, and furosemide all influence transepithelial Na⁺ transport by interacting with the Na⁺ channel or a regulator site of it within the apical membrane. The stoichiometry of the amiloride (RPH 2823)-receptor site interaction revealed Hill-coefficient(s) of less than 1, indicating a negative cooperativity among the receptor sites. The interaction between Na⁺ ions and amiloride or RPH 2823 displayed mixed competitive-noncompetitive inhibition. Taken together, these results support the hypothesis that amiloride and Na⁺ as well as RPH 2823 and Na⁺ may act at different loci on the apical entry mechanism inRana esculenta skin.Abbreviations R Transepithelial electrical resistance - R _x /R₀ Transepithelial electrical resistance at timex (the time after manipulation) divided by the value at time 0 (the time before manipulation) - SCC Short-circuit current - SCC _x /SCC₀ Short-circuit current at timex (the time after manipulation) divided by the value at time 0 (the time before manipulation) Dedicated to Prof. Dr. F. Krück on the occasion of his 65th birthday     

Uncoupling of Na+H+ from Cl−HCO 3 − exchange under some steady state conditions in rabbit gallbladder

The transapical Cl^– influx and transepithelial Na⁺ transport were measured in rabbit gallbladder. Only 11.7% of the transported Na⁺ was found to be accompanied by HCO ₃ ^– . 10^–4 M SITS eliminated the HCO ₃ ^– dependent fraction of Cl^– influx (50%) but did not significantly alter intracellular Na⁺ activity and Na⁺ transport. Exposure to HCO₃-free salines or to 10^–4 M acetazolamide about halved Cl^– influx and Na⁺ transport. 25 mM SCN^– reduced Cl^– influx to zero, decreased intracellular Na⁺ activity, but only halved Na⁺ transport which under these conditions was abolished only in the absence of HCO ₃ ^– . Exposure to a Cl^–-free saline produced effects similar to those caused by SCN^–. These resuits suggest that when Cl^–/HCO ₃ ^– exchange is inhibited at the apical membrane, Na⁺/H⁺ exchange and transepithelial Na⁺ transfer are unmodified if HCO ₃ ^– is available for transport. The permanent uncoupling of the exchangers and the elevated transepithelial transport of Na⁺ are not due to an increased activity of the parallel Na⁺–Cl^– cotransport but to a redirection of HCO ₃ ^– flux toward the basolateral side.     

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.     

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.     

The effect of acute metabolic alkalosis on bicarbonate transport along the loop of Henle. The role of active transport processes and passive paracellular backflux

The loop of Henle (LOH) reabsorbs approximately 15% of filtered HCO ₃ ⁻ via a luminal Na⁺-H⁺ exchanger and H⁺ATPase. During acute metabolic alkalosis (AMA) induced by i.v. HCO ₃ ⁻ infusion, we have observed previously inhibition of LOH net HCO ₃ ⁻ reabsorption , which contributes to urinary elimination of the HCO ₃ ⁻ load and correction of the systemic alkalosis. To determine whether the activities of the Na⁺-H⁺ exchanger and/or H⁺-ATPase are reduced during AMA, two inhibitors believed to be sufficiently specific for each transporter were delivered by in vivo LOH microperfusion during AMA. AMA reduced LOH from 205.0±0.8 to 96.2±11.8 pmol · min⁻¹ (P<0.001). Luminal perfusion with bafilomycin A₁ (10⁻⁴ mol · l⁻¹) caused a further reduction in by 83% and ethylisopropylamiloride (EIPA; 5.10⁻⁴ mol · l⁻¹) completely abolished net HCO ₃ ⁻ reabsorption. The combination of bafilomycin A₁ and EIPA in the luminal perfusate was additive, resulting in net HCO ₃ ⁻ secretion (−66.6±20.8 pmol · min⁻¹;P<0.001) and abolished net fluid reabsorption (from 5.0±0.6 during AMA to 0.2±1.1 nl · min⁻¹;P<0.001). To establish whether HCO ₃ ⁻ secretion via luminal stilbenesensitive transport mechanism participates in LOH adaptation to AMA, we added diisothiocyanato-2,2′-stilbenedisulphonate (DIDS; 10⁻⁴ mol · l⁻¹) to the perfusate. No effect was found. However, when the same LOH were exposed to luminal DIDS for more than 10 min, the direction of net HCO ₃ ⁻ movement was reversed and net HCO ₃ ⁻ secretion occurred: changed from 90.6±8.8 to −91.9±34.1 pmol · min⁻¹;P<0.01, an effect that was not observed in the control state (undisturbed acid-base balance). Thus, during AMA, neither the luminal Na⁺-H⁺ exchanger nor the H⁺-ATPase are noticeably suppressed. However, pharmacological elimination of both transporters, as well as prolonged exposure of the tubular lumen to DIDS, induced net HCO ₃ ⁻ secretion. This secretory flux may reflect paracellular backflux due to the steeper blood to lumen HCO ₃ ⁻ concentration gradient that presumably prevails in AMA.     

Resistive properties of the epithelial membranes of the urinary bladder of the toad,Bufo marinus,determined using the fluorescent dye,RH160

A technique is described for quantitative epifluorescence studies of the apical membrane of the epithelial cells of the urinary bladder of the toad, Bufo marinus, using the lipid-soluble dye, RH160. When the urinary bladder is appropriately mounted, fluorescence signals, in response to a transepithelial voltage pulse, can be recorded from the epithelium immediately after the addition of the dye to the mucosal bath, and for some hours subsequently. The optical signal, recorded as the change in fluorescence in response to a transepithelial voltage pulse, as a fraction of resting fluorescence, was found to be a linear function of the applied voltage over the range ±200 mV, and was approximately 3% for a 100 mV change in transepithelial potential. The signal was enhanced by amiloride (10 μmol · 1⁻¹), reduced by bretylium (5 mmol · 1⁻¹) and abolished in the presence of nystatin (730 U · ml⁻¹). Calculations based on these data permitted estimation of the fractional resistance of the apical membrane, which was found to be 0.85 under control conditions. Apical membrane resistance was 8.6 kΩ · μF, and the basolateral membrane resistance was 1.5 kΩ · μF. These findings support the conclusion that the apical membrane of toad urinary bladder epithelial cells is of high resistance, thus resembling other sodium-transporting epithelia.     

Ion transport and electrophysiology of the early proximal colon of rabbit

The ion transport properties of the mammalian descending colon have been the subject of numerous investigations during the last decade. In contrast, relatively few studies have investigated proximal segments of this organ. In the present study, we assessed transepithelial transport of Na⁺, K⁺ and Cl^– in the isolated initial segment (P1) of rabbit colon in vitro using radioisotopic tracer fluxes and electrophysiological techniques. Like the rabbit descending colon, the proximal colon actively absorbs sodium and chloride, howeveer, its transport systems are markedly different. In vivo, this segment absorbs potassium, however in vitro active potassium secretion was observed. Unlike the descending colon, Na⁺ absorption is relatively insensitive to amiloride and only a slight inhibition was obtained even at 1 mM concentrations of this drug. Na⁺ and Cl^– absorption appeared to be coupled (directly or indicrectly) since the absorption of each ion was inhibited by the removal of the other. Serosal ouabain also inhibited Na⁺ and Cl^– absorption and net K⁺ secretion. Unlike the descending colon, the proximal P1 segment did not have a net absorptive K⁺ transport system that was detectable in the presence of ouabain. Electrically, the early proximal colon has a low transepithelial resistance compared to descending colon (R _T=133±7 cm²) but a larger short-circuit current (l _sc=178±12 A/cm²). The transepithelial potential averaged –21±1 mV, in excellent agreement with values measured in vivo. The apical and basolateral membrane potentials averaged –21±1 mV and –42±1 mV and intracellular potassium activity was 70±2 mM. The findings indicate active K⁺ uptake across the basolateral membrane and passive exit across the apical membrane. The basolateral membrane conductance may be a potassium conductance that is blockable by barium. It is likely that K⁺ transport normally occurs by both cellular and paracellular routes in this epithelium. Because of the numerous differences between this segment and the descending colon, we conclude that the P1 segment of proximal colon has a distinct function in colonic electrolyte transport     

Ionic mechanism of ouabain-induced swelling of leech Retzius neurons

By using electrophysiological and microfluorimetric methods, we found that leech Retzius neurons swell after inhibition of the Na⁺–K⁺ pump by the cardiac glycoside ouabain. To explore the mechanism of this swelling, we measured the effect of ouabain on [Na⁺]_i, [K⁺]_i, and [Cl⁻]_i, as well as on the membrane potential, by applying triple-barrelled ion-sensitive microelectrodes. As shown previously, ouabain induced a marked [Na⁺]_i increase, a [K⁺]_i decrease, and a membrane depolarization, and it also evoked an increase in [Cl⁻]_i. The analysis of the data revealed a net uptake of NaCl, which quantitatively explained the ouabain-induced cell swelling. In the absence of extracellular Na⁺ or Cl⁻, NaCl uptake was excluded, and the cell volume remained unaffected. Likewise, NaCl uptake and, hence, cell swelling did not occur when the Na⁺–K⁺ pump was inhibited by omitting bath K⁺. Also, in K⁺-free solution, [Na⁺]_i increased and [K⁺]_i dropped, but [Cl⁻]_i slightly decreased, and after an initial, small membrane depolarization, the cells hyperpolarized for a prolonged period. It is concluded that the ouabain-induced NaCl uptake is caused by the depolarization of the plasma membrane, which augments the inwardly directed electrochemical Cl⁻ gradient.