Na+ transport by rabbit urinary bladder,a tight epithelium

Summary By in vitro experiments on rabbit bladder, we reassessed the traditional view that mammalian urinary bladder lacks ion transport mechanisms. Since the ratio of actual-to-nominal membrane area in folded epithelia is variable and hard to estimate, we normalized membrane properties to apical membrane capacitance rather than to nominal area (probably 1 F 1 cm² actual area). A new mounting technique that virtually eliminates edge damage yielded resistances up to 78,000 F for rabbit bladder, and resistances for amphibian skin and bladder much higher than those usually reported. This technique made it possible to observe a transport-related conductance pathway, and a close correlation between transepithelial conductance (G) and short-circuit current (I _sc) in these tight epithelia.G andI _sc were increased by mucosal (Na⁺) [I _sc0 when (Na⁺)0], aldosterone, serosal (HCO ₃ ^– ) and high mucosal (H⁺); were decreased by amiloride, mucosal (Ca⁺⁺), ouabain, metabolic inhibitors and serosal (H⁺); and were unaffected by (Cl^–) and little affected by antidiuretic hormone (ADH). Physiological variation in the rabbits' dietary Na⁺ intake caused variations in bladderG andI _sc similar to those caused by the expectedin vivo changes in aldosterone levels. The relation betweenG andI _sc was the same whether defined by diet changes, natural variation among individual rabbits, or most of the above agents. A method was developed for separately resolving conductances of junctions, basolateral cell membrane, and apical cell membrane from thisG–I _sc relation. Net Na⁺ flux equalledI _sc. Net Cl^– flux was zero on short circuit and equalled only 25% of net Na⁺ flux in open circuit. Bladder membrane fragments contained a Na⁺–K⁺-activated, ouabain-inhibited ATPase. The physiological significance of Na⁺ absorption against steep gradients in rabbit bladder may be to maintain kidney-generated ion gradients during bladder storage of urine, especially when the animal is Na⁺-depleted.     

Different modes of electrogenic Na+ absorption in the coprodeum of the chicken embryo: role of extracellular Ca2+

Summary Transepithelial electrogenic Na⁺ transport (I_Na) was investigated in the coprodeum of 20-days-old chicken embryos in Ussing chambers. Short circuit current (I_sc) and transepithelial resistance (R_t) were 14.7±4.8 A · cm^-2 (n=12) and 0.53±0.09 k · cm^-2 (n=12), respectively. I_Na was calculated from changes in I_sc by substitution of mucosal Na⁺ by (N-methyl-d-glucamine) (NMDG). I_sc inversed during Na⁺ removal, and I_Na was found to be 27.8±4.7 A · cm^-2 (n=12). Amiloride (100 mol · l^-1) inhibited only about 60% of I_Na. Analysis of I_sc fluctuations revealed a Lorentzian component in the power density spectrum with a corner frequency of about 57 Hz. This component was not correlated to I_Na, and its origin is still unclear. Removal of mucosal Ca²⁺ increased I_Na about 2.5-fold due to an increase of the amiloride-insensitive component of I_Na in additionally investigated adult tissues. The results clearly show that this is due to a non-selective cation channel with an apparent order of selectivity Cs⁺>Na⁺=K⁺>Rb⁺>Li⁺. The Ca²⁺ concentration required to block 50% of the I_sc was about 18 mol · l^-1. The I _sc ^Ca could also be supressed by other divalent cations such as Mg²⁺ and Ba²⁺. Additionally, an I_Na-linked Lorentzian component occurred which dominated the control spectrum with a significantly higher corner frequency (about 88 Hz). The results indicate that Na⁺ absorption in the coprodeum of the chicken embryo is more complex than in adult hens. However, the Ca²⁺ sensitivity of I_Na is similar to comparable effects described for other epithelia. This possibly reflects the existence of two types of amiloride-insensitive apical cation channels as pathways for Na⁺ absorption, which may be involved to differing degrees in ontogenetic developments of nonselective channels to Na⁺-specific ion channels.Abbreviations DPL direct-linear-plot method - slope of the back-ground noise component - EGTA ethylene glycol-bi(2-amino-ethylether)-N,N,N,N-tetraacetic acid - f frequency - f _c corner frequency of the Lorentzian noise component - G _t transepithelial conductance - HEPES N-hydroxyethylpiperazine-N-ethanesulfonic acid - I _sc short-circuit current - I _Na transepithelial sodium current - I _sc ^Ca Ca²⁺-sensitive short-circuit current - K _m ^Ca Michaelis-Menten constant for Ca²⁺ - K _B power density of the background noise component at f=1Hz - m mucosal - NMDG N-methyl-D-glucamine - R _t transepithelial resistance - s serosal - SEM standard error of mean - S(f) power density of the Lorentzian noise component - S _o plateau value of the Lorentzian noise component     

Effect of aldosterone on ion transport by rabbit colonIn vitro

Summary Segments of descending colon obtained from rabbits, that had been maintained on drinking water containing 25mm NaCl and an artificial diet which contains 1% Na and is nominally K-free, respond to aldosterone in vitro (after a 30 to 60-min lag period) with a marked increase in the short-circuit current (I _sc ), an equivalent increase in the rate of active Na absorption (J _net ^Na ) and a decline in tissue resistance (R _t ). Aldosterone also brings about a marked increase in the unidirectional influx of Na into the cells across the mucosal membrane (zero-time rate of uptake) which does not differ significantly from the increase inI _sc . Treatment of control tissues with amphotericin B brings about sustained increases inI _sc andJ _net ^Na to levels observed in aldosterone-treated tissues. However, addition of amphotericin B to the mucosal solution of aldosteronetreated tissues does not result in a sustained increase inI _sc orJ _net ^Na and these values do not differ markedly from those observed in control tissues treated with amphotericin B. These findings, together with other evidence that Na entry in the presence of amphotericin B is sufficiently rapid to saturate the active Na extrusion mechanism at the baso-lateral membrane, are consistent with the notion that the aldosterone-induced protein increases the permeability of the mucosal membrane to Na but does not increase the saturation level of the active Na pump within the time-frame of these studies (3 hr).Finally, aldosterone has no effect on the bidirectional or net transepithelial movements of K under short-circuit conditions, suggesting that the enhanced secretion of K observed in vivo is the result of increased diffusion of K from plasma to lumen via paracellular pathways in response to an increased transepithelial electrical potential difference (lumen negative).     

The role of the proton electrochemical gradient in the transepithelial absorption of amino acids by human intestinal Caco-2 cell monolayers

We determined the extent of Na⁺-independent, proton-driven amino acid transport in human intestinal epithelia (Caco-2). In Na⁺-free conditions, acidification of the apical medium (apical pH 6.0, basolateral pH 7.4) is associated with a saturable net absorption of glycine. With Na⁺-free media and apical pH set at 6.0, (basolateral pH 7.4), competition studies with glycine indicate that proline, hydroxyproline, sarcosine, betaine, taurine, -alanine, -aminoisobutyric acid (AIB), -methylaminoisobutyric acid (MeAIB), -amino-n-butyric acid and l-alanine are likely substrates for pH-dependent transport in the brush border of Caco-2 cells. Both d-serine and d-alanine were also substrates. In contrast leucine, isoleucine, valine, phenylalanine, methionine, threonine, cysteine, asparagine, glutamine, histidine, arginine, lysine, glutamate and d-aspartate were not effective substrates. Perfusion of those amino acids capable of inhibition of acid-stimulated net glycine transport at the brush-border surface of Caco-2 cell monolayers loaded with the pH-sensitive dye 2,7-bis(2-carboxyethyl-5(6)-carboxyfluorescein) (BCECF) caused cytosolic acidification consistent with proton/amino acid symport. In addition, these amino acids stimulate an inward short-circuit current (I _sc) in voltage-clamped Caco-2 cell monolayers in Na⁺-free media (pH 6.0). Other amino acids such as leucine, isoleucine, phenylalanine, tryptophan, methionine, valine, serine, glutamine, asparagine, d-aspartic acid, glutamic acid, cysteine, lysine, arginine and histidine were without effect on both pH_i and inward I _sc. In conclusion, Caco-2 cells express a Na⁺-independent, H⁺-coupled, rheogenic amino acid transporter at the apical brush-border membrane which plays an important role in the transepithelial transport of a range of amino acids across this human intestinal epithelium.This study was supported by a Wellcome Trust Fellowship (to DTT). Charlotte Ward, Maureen Sinclair and Ken Elliott provided excellent technical assistance.     

Adrenergic regulation of ion transport by primary cultures of canine tracheal epithelium: Cellular electrophysiology

Summary We examined the effect of adrenergic agents on the cellular electrical properties of primary cultures of canine tracheal epithelium. Both isoproterenol and epinephrine stimulated Cl secretion, as evidenced by an increase in transepithelial voltage and a fall in transepithelial resistance. Moreover, both agents appear to increase the conductance of apical and basolateral membranes. However, the pattern of response was different. Isoproterenol initially depolarized apical voltage _a and decreased the fractional resistance of the apical membranef _R. These changes are consistent with an initial increase in apical Cl conductance. In contrast, epinephrine acutely hyperpolarized _a and increasedf _R, changes consistent with an initial increase in basolateral K conductance. Following the acute effect of epinephrine, _a depolarized andf _R decreased to values not significantly different from those observed with isoproterenol. The acute increase in basolateral K conductance produced by epinephrine appeared to result from stimulation of adrenergic receptors because it was reproduced by addition of the agonist phenylephrine, and blocked by the antagonist phentolamine. The ability of prazosin but not yohimbine to block the acute epinephrine-induced increase in K permeability indicates the presence of ₁ adrenergic receptors. The acute adrenergic-induced increase in basolateral K conductance may be mediated by an increase in cell Ca because the response was mimicked by addition of the Ca ionophore A23187. In contrast, the response to isoproterenol was similar to that observed with addition of 8-bromo-cAMP and theophylline. These results indicate that both and adrenergic agents mediate the ion transport processes in canine tracheal epithelium. adrenergic agents have their primary effect on the apical Cl conductance, probably via an increase in cAMP. adrenergic agents exert their primary effect on the basolateral K conductance, possibly via an increase in cell Ca.     

Ion transport across leech integument

The dorsal skin of the leech Hirudo medicinalis was used for electrophysiological measurements performed in Ussing chambers. The leech skin is a tight epithelium (transepithelial resistance = 10.5±0.5 k· cm^-2) with an initial short-circuit current of 29.0±2.9 A·cm^-2. Removal of Na⁺ from the apical bath medium reduced short-circuit current about 55%. Ouabain (50mol·l^-1) added to the basolateral solution, depressed the short-circuit current completely. The Na⁺ current saturated at a concentration of 90 mmol Na⁺·l^-1 in the apical solution (K _M=11.2±1.8 mmol·l^-1). Amiloride (100 mol·l^-1) on the apical side inhibited ca. 40% of the Na⁺ current and indicated the presence of Na⁺ channels. The dependence of Na⁺ current on the amiloride concentration followed Michaclis-Menten kinetics (K _i=2.9±0.4 mol·l^-1). The amiloride analogue benzamil had a higher affinity to the Na⁺ channel (K _i=0.7±0.2 mol·l^-1). Thus, Na⁺ channels in leech integument are less sensitive to amiloride than channels known from vertebrate epithelia. With 20 mmol Na⁺·l^-1 in the mucosal solution the tissue showed an optimum amiloride-inhibitable current, and the amiloride-sensitive current under this condition was 86.8±2.3% of total short-circuit current. Higher Na⁺ concentrations lead to a decrease in amiloride-blockade short-circuit current. Sitmulation of the tissue with cyclic adenosine monophosphate (100 mol·l^-1) and isobutylmethylxanthine (1 mmol·l^-1) nearly doubled short-circuit current and increased amiloride-sensitive Na⁺ currents by 50%. By current fluctuation analysis we estimated single Na⁺ channel current (2.7±0.9 pA) and Na⁺ channel density (3.6±0.6 channels·m^-2) under control conditions. After cyclic adenosine monophosphate stimulation Na⁺ channel density increased to 5.4±1.1 channels·m^-2, whereas single Na⁺ channel current showed no significant change (1.9±0.2 pA). These data present a detailed investigation of an invertebrate epithelial Na⁺ channel, and show the similarities and differences to vertebrate Na⁺ channels. Whereas the channel properties are different from the classical vertebrate Na⁺ channel, the regulation by cyclic adenosine monophosphate seems similar. Stimulation of Na⁺ uptake by cyclic adenosine monophosphate is mediated by an increasing number of Na⁺ channels.Abbreviations slope of the background noise component - ADH antidiuretic hormone - cAMP cyclic adenosine monophosphate - f frequency - f _c coner frequency of the Lorentzian noise component - Hepes N-hydroxyethylpiperazine-N-ethanesulphonic acid - BMX isobutyl-methylxanthine - i _Na single Na⁺ channel current - I _Na max, maximal inhibitable Na⁺ current - I _SC short circuit current - K _i half maximal blocker concentration - K _M Michaelis constandard error of the mean - S _(f) power density of the Lorentzian noise component - S ₀ plateau value of the Lorentzian noise component - TMA tetramethylammonium - Trizma TRIS-hydroxymethyl-amino-methane - V _max maximal reaction velocity - V _T transepithelial potential - K half maximal blocker concentration     

Adrenoceptor heterogeneity in the ruminal epithelium of sheep

The pre-gastric rumen of sheep plays a crucial role in the fermentation of nutrients and in the absorption of nutrients and minerals. Adrenaline has been shown previously to increase ruminal absorption of glucose and water. The present study was intended to elucidate whether ruminal ion transport is also altered by adrenaline. In Ussing chambers, changes of I_sc were recorded in isolated ovine ruminal epithelia after the serosal additions of adrenoceptor agonists or antagonists. I_sc increased after the addition of adrenaline (10^–4 M) or clonidine (₂-agonist, 10^–4 M), but decreased after the addition of isoproterenol (-agonist, 10^–4 M) or terbutaline (₂-agonist, 10^–5 M). The effect of adrenaline on I_sc was augmented by the adrenoceptor antagonists prazosin (₁, 10^–4 M) and bupranolol (, 10^–6 M), but inversed by yohimbine (₂, 10^–5 M). Adrenaline induced an increase in Na⁺ net flux across the epithelium that was larger than the increase in equivalent current flow. It is concluded that adrenaline differentially regulates ion transport across the ruminal epithelium via ₁-, ₂-, and ₂-receptors. The main effect is a stimulation of electroneutral and electrogenic Na⁺ absorption. This stimulated Na⁺ absorption might be causative of increased water absorption from the rumen as described previously.     

Mechanisms of Na and Cl absorption across the distal colon epithelium of the pig

Summary Porcine distal colon epithelium was mounted in Ussing chambers and bathed in plasma-like Ringer solution. Tissue conductances ranged from 10 to 15 mS and the short-circuit current (I_sc) ranged from-15 to 220 A·cm^-2. Variations in basal I_sc resulted from differences in the amount of amiloride (10M mucosal addition)-sensitive Na⁺ absorption. Ion substitution and transepithelial flux experiments showed that 10 M amiloride produced a decrease in the mucosal-to-serosal (M-S) and net Na flux, and that this effect on I_sc was independent of Cl^- and HCO ₃ ^- replacement. When the concentration of mucosal amiloride was increased from 10 to 100 M, little change in I_sc was observed. However, increasing the concentration to 1 mM produced a further inhibition, which often reversed the polarity of the I_sc. The decrease in I_sc due to 1 mM amiloride was dependent on both Cl^- and HCO ₃ ^- , and was attributed to reductions in the M-S and net Na⁺ fluxes as well as the M-S unidirectional Cl^- flux. Ion replacement experiments demonstrated that Cl^- substitution reduced the M-S and net Na fluxes, while replacement of HCO ₃ ^- with HEPES abolished net Cl^- absorption by reducing the M-S unidirectional Cl^- flux. From these data it can be concluded that: (1) Na⁺ absorption is mediated by two distinct amiloride-sensitive transport pathways, and (2) Cl^- absorption is completely HCO ₃ ^- -dependent (presumably mediated by Cl^-/HCO ₃ ^- exchange) and occurs independently of Na⁺ absorption.Abbreviations G_t tissue conductance - HEPES tris (hydroxymethyl) aminomethane - (tris) N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - I_sc short-circuit current - J_r residual flux - M-S mucosal-to-scrosal - S-M serosal-to-mucosal - TTX tetrodotoxin     

The inhibitory chloride channel activated by glutamate as well asγ-amino-butyric acid (GABA)

Summary Outside-out and inside-out patches of membrane were excised from different muscles of crayfish (Austropotamobius torrentium) and single channel currents elicited by synaptic transmitters and their analogues were measured with the patchclamp technique. If the Cl^–-concentration was high on both sides of the membrane, glutamate even at concentrations <1 M elicited low amplitude single channel currents, which were identified to be Cl^–-currents. The same channels were also activated by 10 M GABA. Glutamate and GABA showed competition in activating these inhibitory channels. Amplitude histograms of the single channel currents presented well defined peaks corresponding to 3 channel substatesI ₁,I ₂ andI ₃, with conductances of about(I₁)=22 pS in high chloride corresponding to a permeability _Cl(I₁)=3.5× 10^–14 cm³/s),(I₂)=2(I₁) and(I₃)=3(I₁). Glutamate activated preferably stateI ₁, and GABA stateI ₂, but both could activate all states at sufficient concentration. Distributions of the open times in the different states were plotted and could be fitted each with one or two exponentials described by time constants of(I₁) of 1 and 6 ms,(I₂) of 2 to 3 ms, and(I₃) or 1 to 2 ms. The burst durations had components of 3 to 4 and of 30 to 40 ms. All these durations were approximately the same when the channels were activated by glutamate and GABA. The analogue quisqualate of glutamate, as well as the GABA analogue-guanidino propionic acid also elicited the respective patterns of states of the inhibitory channel. Quisqualate is by far the most effective agonist and glutamate is more effective than GABA at the inhibitory receptor. Picrotoxin blocked activation of the inhibitory channel by GABA more effectively than by glutamate. The importance of the activation of the inhibitory channel by glutamate as well as by GABA and their analogues is discussed. Elements of a tentative reaction schema are proposed.     

Effect of chemical modifiers of amino acid residues on proton conduction by the H+-ATPase of mitochondria

The effect of chemical modifiers of amino acid residues on the proton conductivity of H⁺-ATPase in inside out submitochondrial particles has been studied. Treatment of submitochondrial particles prepared in the presence of EDTA (ESMP) with the arginine modifiers, phenylglyoxal or butanedione, or the tyrosine modifier, tetranitromethane, caused inhibition of the ATPase activity. Phenylglyoxal and tetranitromethane also caused inhibition of the anaerobic release of respiratory H⁺ in ESMP as well as in particles deprived of F₁ (USMP). Butanedione treatment caused, on the contrary, acceleration of anaerobic proton release in both particles. The inhibition of proton release caused by phenylglyoxal and tetranitromethane exhibited in USMP a sigmoidal titration curve. The same inhibitory pattern was observed with oligomycin and withN,N-dicyclohexylcarbodiimide. In ESMP, relaxation of H⁺ exhibited two first-order phases, both an expression of the H⁺ conductivity of the ATPase complex. The rapid phase results from transient enhancement of H⁺ conduction caused by respiratory H⁺ itself. Oligomycin,N,N-dicyclohexylcarbodiimide, and tetranitromethane inhibited both phases of H⁺ release, and butanedione accelerated both. Phenylglyoxal inhibited principally the slow phase of H⁺ conduction. In USMP, H⁺ release followed simple first-order kinetics. Oligomycin depressed H⁺ release, enhanced respiratory H⁺, and restored the biphasicity of H⁺ release. Phenylglyoxal and tetranitromethane inhibited H⁺ release in USMP without modifying its first-order kinetics. Butanedione treatment caused biphasicity of H⁺ release from USMP, introducing a very rapid phase of H⁺ release. Addition of soluble F₁ to USMP also restored biphasicity of H⁺ release. A mechanism of proton conduction by F _o is discussed based on involvement of tyrosine or other hydroxyl residues, in series with the DCCD-reactive acid residue. There are apparently two functionally different species of arginine or other basic residues: those modified by phenylglyoxal, which facilitate H⁺ conduction, and those modified by butanedione, which retard H⁺ diffusion.     

Rotational diffusion tensor of nucleic acids from 13C NMR relaxation

Rotational diffusion properties have been derived for the DNA dodecamer d(CGCGAATTCGCG)₂ from ¹³C R₁ and R₁ measurements on the C₁, C₃, and C₄ carbons in samples uniformly enriched in ¹³C. The narrow range of C-H bond vector orientations relative to the DNA axis make the analysis particularly sensitive to small structural deviations. As a result, the R₁/R₁ ratios are found to fit poorly to the crystal structures of this dodecamer, but well to a recent solution NMR structure, determined in liquid crystalline media, even though globally the structures are quite similar. A fit of the R₁/R₁ ratios to the solution structure is optimal for an axially symmetric rotational diffusion model, with a diffusion anisotropy, D_||/D, of 2.1±0.4, and an overall rotational correlation time, (2D_||+4D)^–1, of 3.35 ns at 35 °C in D₂O, in excellent agreement with values obtained from hydrodynamic modeling.     

Protocol-dependence of equivalent circuit parameters of toad urinary bladder

Summary Determinations of current-voltage relationships are widely employed in the characterization of epithelial sodium transport. In order to determine the protocol dependence of transport parameters in the toad urinary bladder, studies were carried out in the presence and absence of amiloride, an inhibitor of active sodium transport. With symmetric positive and negative perturbations of the transepithelial electrical potential difference (0±100 mV) for 30 sec, the amiloride-sensitive current-voltage (i ^a -) relationship was near linear over the range –75+100 mV, indicating constancy of the conductance ^a and the apparent electromotive force E _Na, lumped parameters of the standard electrical equivalent circuit model of the active transport system. With a reverse protocol (±1000 mV) or 15 min perturbations thei ^a - relationships were highly nonlinear. Nonlinearity reflected voltage dependence of parameters: perturbations that increased active transport decreased E _Na and increased ^a, as evaluated from 10 sec perturbations of ; slowing of active transport produced the converse changes. These effects are usefully analyzed in both quasi-steady states and true steady states by means of a detailed equivalent circuit incorporating the significant ionic currents across each plasma membrane. Precise understanding of the significance of ^a and E _Na will require characterization of the partial ionic conductances on perturbation of .     

Characterization of a Na∶K∶2Cl cotransport system in the apical membrane of a renal epithelial cell line (LLC-PK1)

Summary ⁸⁶Rb uptake into LLC-PK₁ cells (an established renal epithelial cell line) was found to be comprised of an active ouabain-sensitive component, a loop diuretic-sensitive component which was passive and strictly dependent upon the presence of extracellular Na⁺ and Cl^– for activity, and a leak component. The diuretic-sensitive component of influx was investigated further in apical membrane vesicles derived from these cells. A large fraction of⁸⁶Rb,²²Na and³⁶Cl flux into these vesicles was sensitive to inhibition by furosemide and dependent upon the presence of the other two co-ions, in keeping with the presence of a loop diuretic-sensitive Na⁺K⁺Cl^– cotransport system. The kinetic parameters for Na⁺ and K⁺ interaction have been analyzed under initial linear zerotrans conditions. The following values were obtained:K _mNa+=0.42±0.05 mmol/liter,V _max=303±24 pmol/mg/6 sec;K _mK+=11.9±1.0 mmol/liter,V _maxK+=307±27 pmol/mg/6 sec. For Cl^– interaction evidence for two cooperative binding sites with different affinities and different specificities were obtained. Thus, a stoichiometry of 1Na⁺1K⁺2Cl^– can be calculated. It is concluded that the apical membrane of LLC-PK₁ cells contains a Na⁺K⁺2Cl^– cotransport system with properties similar to those described for the thick ascending limb of the loop of Henle.     

Volume-sensitive taurine transport in fish erythrocytes

Summary Taurine plays an important role in cell volume regulation in both vertebrates and invertebrates. Erythrocytes from two euryhaline fish species, the eel (Anguilla japonica) and the starry flounder (Platichthys stellatus) were found to contain high intracellular concentrations of this amino acid ( 30 mmol per liter of cell water). Kinetic studies established that the cells possessed a saturable high-affinity Na⁺-dependent -amino-acid transport system which also required Cl^– for activity (apparentK _m (taurine) 75 and 80 m;V _max 0.85 and 0.29 mol/g Hb per hr for eel (20°C) and flounder cells (10°C), respectively. This -system operated with an apparent Na⁺/Cl^–/taurine coupling ratio of 211. A reduction in extracellular osmolarity, leading to an increase in cell volume, reversibly decreased the activity of the transporter. In contrast, low medium osmolarity stimulated the activity of a Na⁺-independent nonsaturable transport route selective for taurine, -amino-n-butyric acid and small neutral amino acids, producing a net efflux of taurine from the cells. Neither component of taurine transport was detected in human erythrocytes. It is suggested that these functionally distinct transport routes participate in the osmotic regulation of intracellular taurine levels and hence contribute to the homeostatic regulation of cell volume. Volume-induced increases in Na⁺-independent taurine transport activity were suppressed by noradrenaline and 8-bromoadenosine-3, 5-cyclic monophosphate, but unaffected by the anticalmodulin drug, pimozide.     

Studies of sodium channels in rabbit urinary bladder by noise analysis

Summary Sodium channels in rabbit urinary bladder were studied by noise analysis. There are two components of short-circuit current (I _sc) and correspondingly two components of apical Na⁺ entry, one amiloride-sensitive (termedI _A and the A channel, respectively) and one amiloride-insensitive (I _L and the leak pathway, respectively). The leak pathway gives rise tol/f noise, while the A channel in the presence of amiloride gives rise to Lorentzian noise. A two-state model of the A channel accounts well for how the corner frequency and plateau value of Lorentzian noise vary with amiloride concentration. The single-channel current is 0.64 pA, and the conducting channel density is on the order of 40 copies per cell. Triamterene blocks the A channel alone, and increasing external Na⁺ decreases the number but not the single-channel permeability of the A channel. Hydrostatic pressure pulses (punching) increase the number of both pathways. Repeated washing of the mucosal surface removes most of the leak pathway without affecting the A channel.Properties of the A channel revealed by noise analysis of various tight epithelia are compared, and the mechanism ofl/f noise is discussed. It is suggested that the A channel is synthesized intracellularly, stored in intracellular vesicles, transferred with or from vesicular membrane into apical membrane under the action of microfilaments, and degraded into the leak pathway, which is washed out into urine or destroyed. The A channel starts withP _Na/P _K30 and loses selectivity in stages untilP _Na/P _K reaches the free-solution mobility ratio (0.7) for the leak pathway. This turnover cycle functions as a mechanism of repair and regulation for Na⁺ channels, analogous to the repair and regulation of most intracellular proteins by turnover. Vesicular delivery of membrane channels may be operating in several other epithelia.     

Contrasting leaf and ‘ecosystem’ CO2 and H2O exchange in Avena fatua monoculture: Growth at ambient and elevated CO2

Elevated CO₂ (ambient + 35 Pa) increased shoot dry mass production in Avena fatua by 68% at maturity. This increase in shoot biomass was paralleled by an 81% increase in average net CO₂ uptake (A) per unit of leaf area and a 65% increase in average A at the ecosystem level per unit of ground area. Elevated CO₂ also increased ecosystem A per unit of biomass. However, the products of total leaf area and light-saturated leaf A divided by the ground surface area over time appeared to lie on a single response curve for both CO₂ treatments. The approximate slope of the response suggests that the integrated light saturated capacity for leaf photosynthesis is 10-fold greater than the ecosystem rate. Ecosystem respiration (night) per unit of ground area, which includes soil and plant respiration, ranged from-20 (at day 19) to-18 (at day 40) mol m^-2 s^-1 for both elevated and ambient CO₂ Avena. Ecosystem below-ground respiration at the time of seedling emergence was -10 mol m^-2 s^-1, while that occuring after shoot removal at the termination of the experiment ranged from -5 to-6 mol m^-2 s^-1. Hence, no significant differences between elevated and ambient CO₂ treatments were found in any respiration measure on a ground area basis, though ecosystem respiration on a shoot biomass basis was clearly reduced by elevated CO₂. Significant differences existed between leaf and ecosystem water flux. In general, leaf transpiration (E) decreased over the course of the experiment, possibly in response to leaf aging, while ecosystem rates of evapotranspiration (ET) remained constant, probably because falling leaf rates were offset by an increasing total leaf biomass. Transpiration was lower in plants grown at elevated CO₂, though variation was high because of variability in leaf age and ambient light conditions and differences were not significant. In contrast, ecosystem evapotranspiration (ET) was significantly decreased by elevated CO₂ on 5 out of 8 measurement dates. Photosynthetic water use efficiencies (A/E at the leaf level, A/ET at the ecosystem level) were increased by elevated CO₂. Increases were due to both increased A at leaf and ecosystem level and decreased leaf E and ecosystem ET.     

Regulation of NaK-ATPase by platelet-derived growth factors in cultured rat thoracic aortic smooth muscle cells

Regulation of (Na⁺ + K⁺)-adenosine triphosphatase (NaK-ATPase) by platelet-derived growth factor (PDGF) in cultured rat thoracic aortic smooth muscle cells (SMC) was examined. PDGF-BB enhances SMC proliferation and NaK-ATPase activity. Ouabain, an inhibitor of NaK-ATPase activity, prevents PDGF-BB-induced SMC proliferation. As shown by Western blot and immunochemiluminescence analysis, PDGF-BB also enhances ₁, truncated ₁, and ₁ NaK-ATPase subunit levels. PDGF-AA and PDGF-AB show no effect on ₁ and truncated ₁ levels in slot blot analysis. Induction of NaK-ATPase subunit levels by PDGF-BB could be one of the initial events in vascular SMC proliferation.     

Sodium channels contribute to action potential generation in canine and human pancreatic islet B cells

Summary Pancreatic islet B cells depolarize and display trains of action potentials in response to stimulatory concentrations of glucose. Based on data from rodent islets these action potentials are considered to be predominantly Ca²⁺ dependent. Here we describe Na⁺-dependent action potentials and Na⁺ currents recorded from canine and human pancreatic islet B cells. Current-clamp recording using the nystatin perforated-patch technique demonstrates that B cells from both species display tetrodotoxin-sensitive Na⁺ action potentials in response to modest glucose-induced depolarization. In companion whole-cell voltage-clamp experiments on canine B cells, the underlying Na⁺ current displays steep voltage-dependent activation and inactivation over the range of –50 to –40 mV. The Na⁺ current is sensitive to tetrodotoxin block with aK ₁=3.2nm and has a reversal potential which changes with [Na⁺] _o as predicted by the Nernst equation. These results suggest that a voltage-dependent Na⁺ current may contribute significantly to action potential generation in some species outside the rodent family.     

Uptake of lysine and prolinevia separate α-neutral amino acid transport pathways inMytilus gill brush border membranes

Summary Brush border membrane vesicles (BBMV) were prepared from the gills of the marine mussel,Mytilus edulis. These membranes contained two distinct pathways for cotransport of Na⁺ and -neutral amino acids. The major pathway in mussel gill BBMV was the alanine-lysine (AK) pathway, which had a high affinity for alanine and for the cationic amino acid, lysine. The AK pathway was inhibited by nonpolar -neutral amino acids and cationic amino acids, but was not affected by -neutral amino acids or imino acids. The kinetics of lysine transport were consistent with a single saturable process, with aJ _max of 550 pmol/mg-min and aK _t of 5 m. The AK pathway did not have a strict requirement for Na⁺, and concentrative transport of lysine was seen in the presence of inwardly directed gradients of Li⁺ and K⁺, as well as Na⁺. Harmaline inhibited the transport of lysine in solutions containing either Na⁺ or K⁺. The alanine-proline (AP) pathway transported both alanine and proline in mussel gill BBMV. The AP pathway was strongly inhibited by nonpolar -neutral amino acids, proline, and -(methylamino)isobutyric acid (Me-AIB). The kinetics of proline transport were described by a single saturable process, with aJ _max of 180 pmol/mg-min andK _t of 4 m. In contrast to the AK pathway, the AP pathway appeared to have a strict requirement for Na⁺. Na⁺-activation experiments with lysine and proline revealed sigmoid kinetics, indicating that multiple Na⁺ ions are involved in the transport of these substrates. The transport of both lysine and proline was affected by membrane potential in a manner consistent with electrogenic transport.     

Cloning,sequencing and expression of the nhaA and nhaR genes from Salmonella entiritidis

Na⁺/H⁺ antiporter activity is wide-spread and plays essential physiological roles. We found that several Enterobacteriaceae share conserved sequences with nhaA, the gene coding for an E. coli antiporter. A nhaA strain which is sensitive to Na⁺ and Li⁺, was used to clone by complementation a DNA fragment from Salmonella enteritidis which confers resistance to the ions. The cloned fragment increased Na⁺/H⁺ antiport activity in membranes isolated from strains carrying the respective hybrid plasmid. DNA sequence analysis of the insert revealed two open reading frames. Both encode putative polypeptides which are closely homologous to the nhaA and nhaR gene products from Escherichia coli. The antiporter activity displays properties very similar to that of the E. coli NhaA, namely, it is activiated by alkaline pH and recognizes Li⁺ with high affinity.Abbreviations _H ⁺ Proton electrochemical potential - pH transmembrane pH gradient - _Na ⁺ Sodium electrochemical potential - SDS Sodium dodecyl sulfate - CIP Calf intestine alkaline phosphates - ORF open reading frame