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.     

Electrolyte transport across the rabbit caecum in vitro

Electrolyte transport across rabbit caecal epithelium was investigated in vitro using conventional shortcircuiting and radioisotope techniques. In standard saline the caecum exhibited a relatively high short-circuit current (I _sc=4.4 μEq · cm⁻² · h⁻¹) and conductance (6.43 mS · cm⁻²). Both sodium and chloride were absorbed (J _net ^Na =6.40 andJ _net ^Cl =3.40 μEq · cm⁻² · h⁻¹) and potassium was secreted (J _net ^K =−0.5 μEq · cm⁻² · h⁻¹). Removal of Na⁺ abolishedI _sc andJ _net ^Cl whereas removal of Cl⁻ reducedJ _net ^Na to 2.92 μEq · cm⁻² · h⁻¹ but did not alterI _sc. In HCO ₃ ⁻ free salines containing 10⁻⁴ M acetazolamideJ _net ^Cl was abolished andJ _net ^Na andI _sc were reduced to 2.3 and 2.5 μEq · cm⁻² · h⁻¹ respectively. A positive residual ion flux (∼ 1 μEq · cm⁻² · h⁻¹) was detected in standard and Cl⁻-free salines but not in Na⁺-free or HCO ₃ ⁻ buffers. Mucosal amiloride (10⁻³ M) decreased net Na⁺ and Cl⁻ absorption but did not decreaseI _sc. Mucosal DIDS (10⁻⁴ M) decreasedJ _net ^Cl while mucosal bumetanide (10⁻⁴ M) did not affect any of the measured parameters. Finally, addition of theophylline (8 mM) stimulated Cl⁻ secretion and increasedI _sc. It is concluded that net sodium absorption by caecal epithelia occurs by both electrogenic and electroneutral mechanisms whereas net chloride absorption occurs only by an electroneutral process. Coupling of the absorptive fluxes of Na⁺ and Cl⁻ may result from Na⁺/H⁺ and Cl⁻/HCO ₃ ⁻ antiport systems in this tissue. Finally, it is proposed that up to half of theI _sc is due to a Na⁺-dependent secretion of bicarbonate ion.     

Modulation of the transient outward K+ current by inhibition of endothelin-A receptors in normal and hypertrophied rat hearts

Inhibition of endothelin-A (ET_A) receptors has been shown to reduce ventricular electrical abnormalities associated with cardiac failure. In this study, we investigate the effect of ET_A-receptor inhibition on the development of regional alterations of the transient outward K⁺ current (I _to) in the setting of pressure-induced left ventricular (LV) hypertrophy. Cardiac hypertrophy was induced in female Sprague–Dawley rats by stenosis of the ascending aorta (AS) for 7 days. Treatment with the selective ET_A-receptor antagonist darusentan (LU135252, 35 mg [kg body weight]⁻¹ day⁻¹) was started 1 day before the surgery. AS induced a 46% increase in the relative LV weight (p < 0.001) and caused a significant reduction in I _to (at +40 mV) in epicardial myocytes (19.5 ± 1.2 pA pF⁻¹, n = 32 vs 23.2 ± 1.2 pA pF⁻¹, n = 35, p < 0.05). Darusentan further reduced I _to in AS (15.4 ± 1.3 pA pF⁻¹, n = 37, p < 0.05) and sham-operated animals (19.8 ± 1.6 pA pF⁻¹, n = 48, ns.). The effects of AS and darusentan on I _to were significant and independent as tested by two-way analysis of variance. I _to was not affected in endocardial myocytes. These results indicate that endothelin-1 may exert a tonic effect on the magnitude of I _to in the epicardial region of the left ventricle but that ET_A-receptor activation is not necessary for the development of electrical alterations associated with pressure-induced hypertrophy.     

Long-term regulation of vacuolar H+-ATPase by angiotensin II in proximal tubule cells

Long-term effects of angiotensin II (Ang II) on vacuolar H⁺-ATPase were studied in a SV40-transformed cell line derived from rat proximal tubules (IRPTC). Using pH_i measurements with the fluorescent dye BCECF, the hormone increased Na⁺-independent pH recovery rate from an NH₄Cl pulse from 0.066 ± 0.014 pH U/min (n = 7) to 0.14 ± 0.021 pH U/min (n = 13; p < 0.05) in 10 h Ang II (10⁻⁹ M)-treated cells. The increased activity of H⁺-ATPase did not involve changes in mRNA or protein abundance of the B2 subunit but increased cell surface expression of the V-ATPase. Inhibition of tyrosine kinase by genistein blocked Ang II-dependent stimulation of H⁺-ATPase. Inhibition of phosphatidylinositol-3-kinase (PI3K) by wortmannin and of p38 mitogen-activated protein kinase (MAPK) by SB 203580 also blocked this effect. Thus, long-term exposure of IRPTC cells to Ang II causes upregulation of H⁺-ATPase activity due, at least in part, to increased B2 cell surface expression. This regulatory pathway is dependent on mechanisms involving tyrosine kinase, p38 MAPK, and PI3K activation.     

cAMP-dependent inward rectifier current in neurons of the rat suprachiasmatic nucleus

Electrophysiological properties of the inward rectification of neurons in the rat suprachiasmatic nucleus (SCN) were examined by using the single-electrode voltage-clamp method, in vitro. Inward rectifier current (I _H) was produced by hyperpolarizing step command potentials to membrane potentials negative to approximately −60 mV in nominally zero-Ca²⁺ Krebs solution containing tetrodotoxin (1 μM), tetraethylammonium (40 mM), Cd²⁺ (500 μM) and 4-aminopyridine (1 mM).I _H developed during the hyperpolarizing step command potential with a duration of up to 5 s showing no inactivation with time.I _H was selectively blocked by extracellular Cs⁺ (1 mM). The activation of the H-channel conductance (G _H) ranged between −55 and −120 mV. TheG _H was 80–150 pS (n=4) at the half-activation voltage of −84±7 mV (n=4). The reversal potential ofI _H obtained by instantaneous current voltage (I/V) relations was −41±6mV (n=4); it shifted to −51±8mV (n=3) in low-Na⁺ (20 mM) solution and to −24±4 mV (n=4) in high-K⁺ (20 mM) solution. Forskolin (1–10 μM) produced an inward current and increased the amplitude ofI _H. Forskolin did not change the half-activation voltage ofG _H. 8-Bromo-adenosine 3′,5′-cyclic monophosphate (8-Br-cAMP, 0.1–1 mM) and dibutyryl-cAMP (0.1–1 mM) enhancedI _H. 3-Isobutyl-1-methylxanthine (IBMX, 1 mM) also enhancedI _H. The results suggest that the inward rectifier cation current is regulated by the basal activity of adenylate cyclase in neurons of the rat SCN.     

Volume-activated chloride channels in mice Leydig cells

Production and secretion of testosterone in Leydig cells are mainly controlled by the luteinizing hormone (LH). Biochemical evidences suggest that the activity of Cl⁻ ions can modulate the steroidogenic process, but the specific ion channels involved are not known. Here, we extend the characterization of Cl⁻ channels in mice Leydig cells (50–60 days old) by describing volume-activated Cl⁻ currents (I_Cl,swell). The amplitude of I_Cl,swell is dependent on the osmotic gradient across the cell membrane, with an apparent EC₅₀ of ∼75 mOsm. These currents display the typical biophysical signature of volume-activated anion channels (VRAC): dependence on intracellular ATP, outward rectification, inactivation at positive potentials, and selectivity sequence (I⁻> Cl⁻> F⁻). Staurosporine (200 nM) did not block the activation of I_Cl,swell. The block induced by 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB; 128 μM), SITS (200 μM), ATP (500 μM), pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonate (PPADS; 100 μM), and Suramin (10 μM) were described by the permeant blocker model with apparent dissociation constant at 0 mV and fractional distance of the binding site (δ) of 334 μM and 47 %, 880 μM and 35 %, 2,100 μM and 49%, 188 μM and 27%, and 66.5 μM and 49%, respectively. These numbers were derived from the peak value of the currents. We conclude that I_Cl,swell in Leydig cells are activated independently of purinergic stimulation, that Suramin and PPADS block these currents by a direct interaction with VRAC and that ATP is able to permeate this channel.     

The rise of [Na+]i during ischemia and reperfusion in the rat heart—underlying mechanisms

Intracellular Na⁺ concentration ([Na⁺]_i) rises in the heart during ischemia, and on reperfusion, there is a transient rise followed by a return toward control. These changes in [Na⁺]_i contribute to ischemic and reperfusion damage through their effects on Ca²⁺ overload. Part of the rise of [Na⁺]_i during ischemia may be caused by increased activity of the cardiac Na⁺/H⁺ exchanger (NHE1), activated by the ischemic rise in [H⁺]_i. In support of this view, NHE1 inhibitors reduce the [Na⁺]_i rise during ischemia. Another possibility is that the rise of [Na⁺]_i during ischemia is caused by Na⁺ influx through channels. We have reexamined these issues by use of two different NHE1 inhibitors, amiloride, and zoniporide, in addition to tetrodotoxin (TTX), which blocks voltage-sensitive Na⁺ channels. All three drugs produced cardioprotection after ischemia, but amiloride (100 μM) and TTX (300 nM) prevented the rise in [Na⁺]_i during ischemia, whereas zoniporide (100 nM) did not. Both amiloride and zoniporide prevented the rise of [Na⁺]_i on reperfusion, whereas TTX was without effect. In an attempt to explain these differences, we measured the ability of the three drugs to block Na⁺ currents. At the concentrations used, TTX reduced the transient Na⁺ current (I _Na) by 11 ± 2% while amiloride and zoniporide were without effect. In contrast, TTX largely eliminated the persistent Na⁺ current (I _Na,P) and amiloride was equally effective, whereas zoniporide had a substantially smaller effect reducing I _Na,P to 41 ± 8%. These results suggest that part of the effect of NHE1 inhibitors on the [Na⁺]_i during ischemia is by blockade of I _Na,P. The fact that a low concentration of TTX eliminated the rise of [Na⁺]_i during ischemia suggests that I _Na,P is a major source of Na⁺ influx in this model of ischemia.     

Trisk 32 regulates IP(3) receptors in rat skeletal myoblasts

To date, four isoforms of triadins have been identified in rat skeletal muscle. While the function of the 95-kDa isoform in excitation–contraction coupling has been studied in detail, the role of the 32-kDa isoform (Trisk 32) remains elusive. Here, Trisk 32 overexpression was carried out by stable transfection in L6.G8 myoblasts. Co-localization of Trisk 32 and IP₃ receptors (IP₃R) was demonstrated by immunocytochemistry, and their association was shown by co-immunoprecipitation. Functional effects of Trisk 32 on IP₃-mediated Ca²⁺ release were assessed by measuring changes in [Ca²⁺]_i following the stimulation by bradykinin or vasopressin. The amplitude of the Ca²⁺ transients evoked by 20 μM bradykinin was significantly higher in Trisk 32-overexpressing (p < 0.01; 426 ± 84 nM, n = 27) as compared to control cells (76 ± 12 nM, n = 23). The difference remained significant (p < 0.02; 217 ± 41 nM, n = 21, and 97 ± 29 nM, n = 31, respectively) in the absence of extracellular Ca²⁺. Similar observations were made when 0.1 μM vasopressin was used to initiate Ca²⁺ release. Possible involvement of the ryanodine receptors (RyR) in these processes was excluded, after functional and biochemical experiments. Furthermore, Trisk 32 overexpression had no effect on store-operated Ca²⁺ entry, despite a decrease in the expression of STIM1. These results suggest that neither the increased activity of RyR, nor the amplification of SOCE, is responsible for the differences observed in bradykinin- or vasopressin-evoked Ca²⁺ transients; rather, they were due to the enhanced activity of IP₃R. Thus, Trisk 32 not only co-localizes with, but directly contributes to, the regulation of Ca²⁺ release via IP₃R.     

Interaction of gonadal steroids and the glucocorticoid corticosterone in the regulation of the L-type Ca(2+) current in rat left ventricular cardiomyocytes

Aim: Gonadal steroids as well as glucocorticoids have been shown to regulate the cardiac L‐type Ca²⁺ current (I_CaL). Herein, we compare the effects of the gonadal steroids testosterone and 17β‐estradiol with the glucocorticoid corticosterone on I_CaL, and investigate the interaction between the gonadal steroids and corticosterone. Methods: Myocytes were isolated from the left ventricular free wall of female and male Wistar rats and investigated using the ruptured‐patch whole‐cell patch‐clamp technique. Results: In myocytes isolated from female rats, 24 h incubation with 100 nm testosterone led to a 33% increase in I_CaL compared with control (?8.8 ± 0.5 pA pF^?1, n = 25 vs. ?6.6 ± 0.4 pA pF^?1, n = 26, P < 0.01, V_Pip = 0 mV). Incubation with 1 μm corticosterone resulted in a 79% increase in I_CaL (?11.8 ± 0.7 pA pF^?1, n = 29, P < 0.001). However, the combination of testosterone and corticosterone did not have any additional effect compared with corticosterone alone (?11.7 ± 0.6 pA pF^?1, n = 25, ns). In cardiomyocytes from male rats, I_CaL was not affected by testosterone, whereas the effect of corticosterone was preserved (P < 0.05). 24 h incubation with 17β‐estradiol increased I_CaL by 32% from ?7.6 ± 0.5 pA pF^?1 (n = 15) to 10.0 ± 0.9 pA pF^?1 (n = 15, P < 0.05). 17β‐estradiol did not exert an additional effect upon co‐incubation with corticosterone and did not have an effect on I_CaL in cardiomyocytes from female rats. Higher concentrations of the gonadal steroids did not result in increased effects. Conclusion: When compared with corticosterone, the in vitro effects of the gonadal steroids are small. However, under conditions in which I_CaL is not fully activated by glucocorticoids, gonadal steroids may significantly contribute to I_CaL regulation.     

Late Na+ current produced by human cardiac Na+ channel isoform Nav1.5 is modulated by its β1 subunit

Experimental data accumulated over the past decade show the emerging importance of the late sodium current (I _NaL) for the function of both normal and, especially, failing myocardium, in which I _NaL is reportedly increased. While recent molecular studies identified the cardiac Na⁺ channel (NaCh) α subunit isoform (Na_v1.5) as a major contributor to I _NaL, the molecular mechanisms underlying alterations of I _NaL in heart failure (HF) are still unknown. Here we tested the hypothesis that I _NaL is modulated by the NaCh auxiliary β subunits. tsA201 cells were transfected simultaneously with human Na_v1.5 (former hH1a) and cardiac β₁ or β₂ subunits, and whole-cell patch-clamp experiments were performed. We found that I _NaL decay kinetics were significantly slower in cells expressing α + β₁ (time constant τ = 0.73 ± 0.16 s, n = 14, mean ± SEM, P < 0.05) but remained unchanged in cells expressing α + β₂ (τ = 0.52 ± 0.09 s, n = 5), compared with cells expressing Na_v1.5 alone (τ = 0.54 ± 0.09 s, n = 20). Also, β₁, but not β₂, dramatically increased I _NaL relative to the maximum peak current, I _NaT (2.3 ± 0.48%, n = 14 vs. 0.48 ± 0.07%, n = 6, P < 0.05, respectively) and produced a rightward shift of the steady-state availability curve. We conclude that the auxiliary β₁ subunit modulates I _NaL, produced by the human cardiac Na⁺ channel Na_v1.5 by slowing its decay and increasing I _NaL amplitude relative to I _NaT. Because expression of Na_v1.5 reportedly decreases but β₁ remains unchanged in chronic HF, the relatively higher expression of β₁ may contribute to the known I _NaL increase in HF via the modulation mechanism found in this study. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

HCO 3 − -dependent volume regulation in α-cells of the rat endocrine pancreas

Ion transport activity in pancreatic α-cells was assessed by studying cell volume regulation in response to anisotonic solutions. Cell volume was measured by a video imaging method, and cells were superfused with either 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid-buffered or HCO₃⁻-buffered solutions. α-Cells did not exhibit a regulatory volume increase (RVI) in response to cell shrinkage caused by hypertonic solutions. A RVI was observed, however, in cells that had first undergone a regulatory volume decrease (RVD), but only in HCO₃⁻-buffered solutions. RVI was also observed in response to a HCO₃⁻-buffered hypertonic solution in which the glucose concentration was increased from 4 to 20 mM. The post-RVD RVI and the glucose-induced RVI were both inhibited by 10 μM 5-(N-methyl-N-isobutyl) amiloride or 100 μM 2,2′-(1,2-ethenediyl) bis (5-isothio-cyanatobenzenesulfonic acid), but not by 10 μM benzamil nor 10 μM bumetanide. These data suggest that Na⁺–H⁺ exchangers and Cl⁻–HCO₃⁻ exchangers contribute to volume regulation in α-cells.     

Effects of K-201 on the calcium pump and calcium release channel of rat skeletal muscle

The benzothiazepine derivative K-201 has been suggested as a potential therapeutic agent due to its antiarrhythmogenic action. To understand how the drug alters calcium release from the sarcoplasmic reticulum (SR), we investigated its effects on the SR calcium channel and calcium pump by single channel electrophysiology, whole-cell confocal microscopy, and ATPase activity measurements on control and post-myocardial infarcted (PMI) rat skeletal muscle. In bilayers, K-201 induced two subconductance states corresponding to ∼24% (S₁) and ∼13% (S₂) of the maximum conductance. Dependence of event frequency and of time spent in S₁ and S₂ on the drug concentration was biphasic both in control and in PMI rats, with a maximum at 50 μM. At this concentration, the channel spends 26 ± 4% and 24 ± 4%, respectively, of the total time in these subconductance states at positive potentials, while no subconductances are observed at negative potentials. K-201 altered the frequency of elementary calcium release events: spark frequency decreased from 0.039 ± 0.001 to 0.023 ± 0.001 s⁻¹ sarcomere⁻¹, while the frequency of embers increased from 0.011 ± 0.001 to 0.023 ± 0.001 s⁻¹ sarcomere⁻¹. Embers with different amplitude levels were observed after the addition of the drug. K-201 inhibited the Ca²⁺ ATPase characterized by IC_50,contr = 119 ± 21 μM and n _Hill,contr = 1.84 ± 0.48 for control and IC_50,PMI = 122 ± 18 μM and n _Hill,PMI = 1.97 ± 0.24 for PMI animals. These results suggest that although K-201 would increase the appearance of subconductance states, the overall calcium release is reduced by the drug. In addition, the effect of K-201 is identical on calcium release channels from control and PMI rats.     

Evidence for a Na+/Ca2+ exchange mechanism in frog skin epithelium

 In the present study we investigated the possible existence of a Na⁺/Ca²⁺ exchange mechanism in the basolateral membrane of the frog skin epithelium and whether such a mechanism plays a role in the regulation of transepithelial Na⁺ transport. Cytosolic calcium ([Ca²⁺]_i) was measured with the probe fura-2 in a set-up in which pieces of tissue were mounted on the stage of an epifluorescence microscope. Na⁺ transport was measured as the amiloride-sensitive short-circuit current (I _sc) using a conventional voltage clamp. Basal [Ca²⁺]_i was 65±6 nM (n=15). Removal of Na⁺ from the mucosal solution had no effect on [Ca²⁺]_i. When Na⁺ was removed from the serosal solution, [Ca²⁺]_i increased biphasically to a peak of 220±38 nM (n=8, P=0.006). Readdition of Na⁺ to the serosal solution returned [Ca²⁺]_i to control level. The serosal Na⁺ gradient and changes in [Ca²⁺]_i were closely correlated; stepwise changes in serosal Na⁺ were followed by stepwise changes in [Ca²⁺]_i. These observations indicate the existence of a Na⁺/Ca²⁺ exchange mechanism in the basolateral membrane of the frog skin epithelium. The transepithelial Na⁺ transport decreased from 13.2±1.8 to 9.2±1.5 μA cm^–2 (n=8, P=0.049) when Na⁺ was omitted from the serosal solution. When this protocol was repeated in the absence of serosal Ca²⁺, Na⁺ transport decreased similarly from 16.7±1.7 to 11.6 ±1.8 μA cm^–2 (n=6, P=0.004). We conclude that it is unlikely that the observed decrease in I _sc after removal of serosal Na⁺ is due to an increase in [Ca²⁺]_i per se. Received: 10 July 1998 / Received after revision: 23 September 1998 / Accepted: 25 September 1998     

Persistent sodium current and Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchange contributes to the augmentation of the reverse Na<Superscript>+</Superscript>/Ca<Superscript>2+</Superscript> exchange during hypoxia or acute ischemia in ventricular myocytes

The increases in persistent sodium currents (I _Na.P) and Na⁺/H⁺ exchange (NHE) causes intracellular Ca²⁺ overload. The objective of this study was to determine the contribution of I _Na.P and NHE on the hypoxia- or acute ischemia-induced increase in the reverse Na⁺/Ca²⁺ exchange current (HIR- or AIR-I _NCX). I _Na.P and I _NCX in rabbit ventricular myocytes were recorded during hypoxia or acute ischemia, combination of acidosis (pH values were 6.0 intracellularly and 6.8 extracellularly) and hypoxia, using whole-cell patch-clamp techniques. The results indicate that (1) under hypoxic condition, the augmentation of both HIR-I _NCX and I _Na.P was inhibited by TTX (2 to 8 μM) in a concentration-dependent manner. The inhibitions of I _Na,P and HIR-I _NCX reached maximum in the presence of either 4 μM TTX or 10 μM KR-32568 (a NHE inhibitor), respectively. The maximal inhibitions of HIR-I _NCX by 4 μM TTX and 10 μM KR-32568 were 72.54% and 16.89%, respectively. (2) Administration of 2 μM TTX and 10 μM KR-32568 in either order in the same cells decreased HIR-I _NCX by 64.83% and 16.94%, respectively. (3) I _Na.P and the reverse I _NCX were augmented during acute ischemia. TTX (4 μM) and KR-32568 (10 μM) reduced AIR-I _NCX by 73.39% and 24.13%, respectively. (4) Under normoxic condition, veratridine (20 μM) significantly increased I _Na.P and the reverse I _NCX, which was reversed by 4 μM TTX. In conclusion, during hypoxia or acute ischemia, both increased I _Na.P and NHE contribute to the HIR- or AIR-I _NCX with the former playing a major role comparing with the latter.     

Higher prevalence of exercise-associated hyponatremia in female than in male open-water ultra-endurance swimmers: the 'Marathon-Swim' in Lake Zurich

We investigated the prevalence of exercise-associated hyponatremia (EAH) in 25 male and 11 female open-water ultra-endurance swimmers participating in the ‘Marathon-Swim’ in Lake Zurich, Switzerland, covering a distance of 26.4 km. Changes in body mass, fat mass, skeletal muscle mass, total body water, urine specific gravity, plasma sodium concentration [Na⁺] and haematocrit were determined. Two males (8%) and four females (36%) developed EAH where one female was symptomatic with plasma sodium [Na⁺] of 127 mmol/L. Body mass and plasma [Na⁺] decreased (p < 0.05). The changes in body mass correlated in both male and female swimmers to post-race plasma [Na⁺] (r = −0.67, p = 0.0002 and r = −0.80, p = 0.0034, respectively) and changes in plasma [Na⁺] (r = −0.68, p = 0.0002 and r = −0.79, p = 0.0039, respectively). Fluid intake was neither associated with changes in body mass, post-race plasma [Na⁺] or the change in plasma [Na⁺]. Sodium intake showed no association with either the changes in plasma [Na⁺] or post-race plasma [Na⁺]. We concluded that the prevalence of EAH was greater in female than in male open-water ultra-endurance swimmers.     

Properties and functional implications of <Emphasis Type="Italic">I</Emphasis><Subscript>h</Subscript> in hippocampal area CA3 interneurons

The present study examines the biophysical properties and functional implications of I _h in hippocampal area CA3 interneurons with somata in strata radiatum and lacunosum-moleculare. Characterization studies showed a small maximum h-conductance (2.6 ± 0.3 nS, n = 11), shallow voltage dependence with a hyperpolarized half-maximal activation (V _1/2 = −91 mV), and kinetics characterized by double-exponential functions. The functional consequences of I _h were examined with regard to temporal summation and impedance measurements. For temporal summation experiments, 5-pulse mossy fiber input trains were activated. Blocking I _h with 50 μM ZD7288 resulted in an increase in temporal summation, suggesting that I _h supports sensitivity of response amplitude to relative input timing. Impedance was assessed by applying sinusoidal current commands. From impedance measurements, we found that I _h did not confer theta-band resonance, but flattened the impedance–frequency relations instead. Double immunolabeling for hyperpolarization-activated cyclic nucleotide-gated proteins and glutamate decarboxylase 67 suggests that all four subunits are present in GABAergic interneurons from the strata considered for electrophysiological studies. Finally, a model of I _h was employed in computational analyses to confirm and elaborate upon the contributions of I _h to impedance and temporal summation.     

Volume-activated,gadolinium-sensitive whole-cell currents in single proximal cells of frog kidney

Stretch-activated channels (SACs) have been implicated in the control of epithelial cell volume. Such channels are generally sensitive to the trivalent lanthanide, gadolinium (Gd³⁺). In this study, using Gd³⁺ sensitivity and volume activation as indices, we have looked for ionic currents attributable to SACs using the wholecell-patch clamp technique in freshly isolated proximal tubule cells of the frog. Hypotonic shock caused a reversible increase in whole-cell conductance, which was inhibited by Gd³⁺. In conjunction with this increase in conductance, cell length (measured using an optical technique) also increased. We observed two types of volume and Gd³⁺-sensitive currents: voltage-dependentI _VD and voltage-independent I_VI. I_VD was found in all cells, activated by depolarisation and hypotonic shock, and was inhibited reversibly by 10 μM Gd³⁺. The conductance did not discriminate between Na⁺ and K⁺ but was slightly anion-selective and was Ca²⁺-permeable.I _VI was observed in only 50% of cells and was also inhibited by Gd³⁺. Although the inhibition was irreversible, it was dose-dependent, suggesting a specific effect of Gd³⁺ onI _VI. Cells that showed I_VI had a significantly higher conductance than those that did not (38.7±4.4,n=20, and 20.5±0.7,n=15, μS · cm⁻² respectively). In contrast toI _VD,I _VI was mildly cation-selective, Ca²⁺-permeable, and also selective for Na⁺ over K⁺. As withI _VD, volume-induced increases inI _VI were inhibited by Gd³⁺. Both of these currents are activated during hypotonic shock and may be involved in volume-regulatory processes in frog proximal cells. This work was supported by the Wellcome Trust     

PI3-Kinase-dependent electrogenic intestinal transport of glucose and amino acids

Intestinal glucose and amino acid transport is stimulated by the serum- and glucocorticoid-inducible kinase isoforms SGK1, SGK2, and SGK3 and protein kinase B which are, in turn, stimulated following activation of the phosphoinositol-3 kinase (PI3 kinase). The present study has been performed to explore whether pharmacological inhibition of the PI3 kinase affects electrogenic jejunal transport of glucose and amino acids. In Ussing chamber experiments, glucose (20 mM), phenylalanine (20 mM), glutamine (20 mM), cysteine (20 mM), and proline (20 mM) generated lumen negative currents (I _glc, I _phe, I _gln, I _cys, and I _pro), respectively, which gradually declined following application of the PI3 kinase inhibitor Wortmannin (1 μM). Within 40 min, Wortmannin treatment significantly decreased I _glc by 39 ± 10% (n = 5), I _phe by 70 ± 7% (n = 4), I _gln by 69 ± 8% (n = 4), I _cys by 67 ± 8% (n = 6), and I _prol by 79 ± 12% (n = 3). A similar decline of I _glc was observed following application of the PI3 kinase inhibitor LY294002 (50 μM). Exposure to the inhibitors did not significantly alter transepithelial potential difference and resistance in the absence of substrates for electrogenic transport. The observations suggest that the electrogenic transport of glucose and several amino acids requires the continued activity of PI3 kinase. R. Rexhepaj and F. Artunc shared first authorship.     

Sweat sodium concentration during exercise in the heat in aerobically trained and untrained humans

The purpose of this study was to determine whether sweat sodium concentration ([Na⁺]_sweat) during exercise in the heat differs between aerobically trained and untrained individuals. On three occasions, ten endurance-trained (Tr) and ten untrained (UTr) subjects ( [(V)\dot]\textO_2\textpeak \dot{V}{\text{O}}_{{2{\text{peak}}}}  = 4.0 ± 0.8 vs. 3.4 ± 0.7 L min⁻¹, respectively; P < 0.05) cycled in a hot-ventilated environment (36 ± 1°C; 25 ± 2% humidity, airflow 2.5 m s⁻¹) at three workloads (i.e., 40, 60, and 80% [(V)\dot]\textO_2\textpeak \dot{V}{\text{O}}_{{2{\text{peak}}}} ). Whole-body (SR_WB) and back sweat rates (SR_BACK) were measured. At the conclusion of the study, Na⁺ in sweat and blood samples was analyzed to calculate Na⁺ secretion and reabsorption rates. SR_WB and SR_BACK were highly correlated in Tr and UTr (r = 0.74 and 0.79, respectively; P < 0.0001). In both groups, SR_BACK increased with the increases in exercise intensity (P < 0.05). Likewise, [Na⁺]_sweat increased with the exercise intensity in both groups (P < 0.05) and it tended to be higher in Tr than in UTr at 60 and 80% [(V)\dot]\textO_2\textpeak \dot{V}{\text{O}}_{{2{\text{peak}}}} (~22 mmol L⁻¹ higher; P = 0.06). However, when normalized for SR_BACK, [Na⁺]_sweat was not different between groups. In both groups, Na⁺ secretion and reabsorption rates increased with the increases in SR_BACK (P < 0.05). However, Na⁺ reabsorption rate was lower in the Tr than in the UTr (mean slope = 48 vs. 82 ηmol cm⁻² min⁻¹; P = 0.03). In conclusion, using a cross-sectional study design, our data suggest that aerobic fitness level does not reduce sweat Na⁺ secretion or enhance Na⁺ reabsorption during prolonged exercise in the heat that induced high sweat rates.     

Contribution of BK channels to action potential repolarisation at minimal cytosolic Ca2+ concentration in chromaffin cells

BK channels modulate cell firing in excitable cells in a voltage-dependent manner regulated by fluctuations in free cytosolic Ca²⁺ during action potentials. Indeed, Ca²⁺-independent BK channel activity has ordinarily been considered not relevant for the physiological behaviour of excitable cells. We employed the patch-clamp technique and selective BK channel blockers to record K⁺ currents from bovine chromaffin cells at minimal intracellular (about 10 nM) and extracellular (free Ca²⁺) Ca²⁺ concentrations. Despite their low open probability under these conditions (V₅₀ of +146.8 mV), BK channels were responsible for more than 25% of the total K⁺ efflux during the first millisecond of a step depolarisation to +20 mV. Moreover, BK channels activated about 30% faster (τ = 0.55 ms) than the rest of available K⁺ channels. The other main source of fast voltage-dependent K⁺ efflux at such a low Ca²⁺ was a transient K⁺ (I_A-type) current activating with V ₅₀ = −14.2 mV. We also studied the activation of BK currents in response to action potential waveforms and their contribution to shaping action potentials both in the presence and the absence of extracellular Ca²⁺. Our results show that BK channels activate during action potentials and accelerate cell repolarisation even at minimal Ca²⁺ concentration, and suggest that they could do so also in the presence of extracellular Ca²⁺, before Ca²⁺ entering the cell facilitates their activity.