Low-amiloride-affinity Na+ channel in the epithelial cells isolated from the endolymphatic sac of guinea-pigs

 By using the whole-cell patch-clamp technique, an amiloride-sensitive Na⁺-selective conductance was found in epithelial cells from the endolymphatic sac (ES) epithelia of guinea-pigs. In the current-clamp configuration, the average resting membrane potential was –41.7±8.4 mV (n = 22). Application of amiloride at a concentration of 20 μM elicited a decrease in cation conductance that was responsible for a membrane hyperpolarization by 17.9±6.0 mV (n = 22). Substitution of N-methyl d-glucamine chloride (NMDG-Cl) for external NaCl led to a more significant membrane hyperpolarization by 28.4±8.3 mV (n = 22). At holding potential of –70 mV, amiloride and ethylisopropylamiloride (EIPA) blocked the inward current in a concentration-dependent manner over the range of concentrations of between 0.1 μM and 50 μM, with an inhibitory constant (K _i) of 1.3±0.4 μM (n = 7) and 1.5±0.3 μM (n = 5), respectively. In the voltage-clamp configuration, substitution of NMDG-Cl for external NaCl significantly reduced the inward current (n = 9), indicating that the whole-cell conductance has a high permeability for Na⁺. Superfusion with 20 μM amiloride induced a significant reduction of the inward current, shifted the reversal potential from –39.4±8.8 mV to –60.4±10.5 mV (n = 12), and decreased the inward conductance from 5.0±1.3 nS to 3.7±1.5 nS (n = 12). The permeability ratio of Na⁺ over K⁺, calculated from the difference in reversal potential between the currents before and after application of amiloride, was approximately 5:1. Additionally, the conductance was not activated by application of forskolin, 3-isobutyl-1-methylxanthine (IBMX) and 8-bromo-cAMP (8-Br-cAMP). These findings suggest that a low-amiloride-affinity Na⁺ channel localized in the ES epithelial cells may be involved in uptake of Na⁺ in the ES. Received: 29 May 1996 / Received after revision: 1 August 1996 / Accepted: 2 August 1996     

Activation of PKC increases Na+-K+ pump current in ventricular myocytes from guinea pig heart

 We have previously shown activation of α₁-adrenergic receptors increases Na⁺-K⁺ pump current (I _p) in guinea pig ventricular myocytes, and the increase is eliminated by blockers of phosphokinase C (PKC). In this study we examined the effect of activators of PKC on I _p. Phorbol 12-myristate 13-acetate (PMA), a PKC activator, increased I _P at each test potential without shifting its voltage dependence. The concentration required for a half-maximal response (K _0.5) was 6 μM at 15 nM cytosolic [Ca²⁺] ([Ca²⁺]_i) and13 nM at 314 nM [Ca²⁺]_i. The maximal increase at either [Ca²⁺]_i was about 30%. Another activator of PKC, 1,2-dioctanoyl-sn-glycerol (diC₈), increased I _p similarly. The effect of PMA on I _P was eliminated by the PKC inhibitor staurosporine, but not by the peptide PKI, an inhibitor of protein kinase A (PKA). PMA and α₁-adrenergic agonist effects both were sensitive to [Ca²⁺]_i, blocked by PKC inhibitors, unaffected by PKA inhibition, and increased I _p uniformly at all voltages. However, they differed in that α₁-activation caused a maximum increase of 15% vs 30% via PMA, and α₁-effects were less sensitive to [Ca²⁺]_i than PMA effects. These results demonstrate that activation of PKC causes an increase in I _p in guinea pig ventricular myocytes. Moreover, they suggest that the coupling of α₁-adrenergic activation to I _p is entirely through PKC, however α₁-activation may be coupled to a specific population of PKC whereas PMA is a more global agonist. Received: 21 August 1998 / Received after revision: 7 December 1998 / Accepted: 11 December 1998     

Intracellular Na+ measurements in smooth muscle using SBFI – changes in [Na+], Ca2+ and force in normal and Na+-loaded ureter

 Our understanding of the control and effects of intracellular [Na⁺] ([Na⁺]_i) in intact smooth muscle is limited by the lack of data concerning [Na⁺]_i. The initial aim of this work was therefore to investigate the suitability of using the Na⁺-sensitive fluorophore SBFI in intact smooth muscle. We find this to be a good method for measuring [Na⁺]_i in ureteric smooth muscle. Resting [Na⁺]_i was found to be around 10 mM and rose to 25 mM when the Na⁺-K⁺-ATPase was inhibited by ouabain. This relatively low [Na⁺]_i in the absence of Na⁺-K⁺-ATPase suggests that other cellular processes, such as Na⁺-Ca²⁺ exchange, play a role in maintaining [Na⁺]_i under these conditions. Simultaneous measurements of [Na⁺]_i or [Ca²⁺] _i and force showed that Na⁺-Ca²⁺ exchange can play a functional role in ureteric smooth muscle. We found that the greater the driving force for Na⁺ exit and hence Ca²⁺ entry, the larger the contraction. In addition the Na⁺-Ca²⁺ exchanger activity under these conditions was found to be pH sensitive: acidification reduced the contraction and concomitant changes in [Ca²⁺] and [Na⁺]_i. We conclude that SBFI is a useful method for monitoring [Na] in smooth muscle and that Na⁺-Ca²⁺ exchange may play a functional role in the ureter. Received: 26 August 1997 / Received after revision: 27 October 1997 / Accepted: 28 October 1997     

Amiloride-inhibitable Na+ conductance in rat proximal tubule

 Previous single-channel recordings from the luminal membrane of the rabbit proximal tubule have revealed amiloride-inhibitable Na⁺ channels of a characteristic conductance range. The present study aimed to pursue this issue in rat proximal tubule. Control rats were compared to those put on a low-Na⁺ diet or pretreated by triamcinolone injections (s.c.). Stimulation of Na⁺ absorption by glucocorticoids was verified by examining the transepithelial voltage in Ussing chamber studies of the distal colon. The membrane voltage (V _m) of isolated, in-vitro-perfused proximal tubule segments was measured in patch-clamp and impalement studies. It was found that amiloride hyperpolarized V _m significantly by 2.1 ± 0.9 mV (n = 26) in tubules of control rats, by 3.9 ± 0.7 mV (n = 12) in rats put on a low-Na⁺ diet and by 3.7 ± 1.0 mV (n = 17) in rats treated with glucocorticoids. The effect of amiloride was concentration dependent with a half-maximal effect at < 1 μmol/l. RT-PCR techniques were used to search for the presence of the α-, β- and γ-subunits of the epithelial Na⁺ channel in isolated oximal tubule segments. The presence of the respective mRNAs was verified. These data indicate that: (1) amiloride-inhibitable Na⁺ channels are present in rat proximal tubules; (2) the Na⁺ conductance may be upregulated by Na⁺ deprivation but is still very limited when compared to total cell conductance; (3) therefore, the contribution of Na⁺-channel-mediated absorption to total proximal Na⁺ absorption is probably small. Received: 5 August 1996 / Received after revision: 22 January 1997 / Accepted: 28 January 1997     

Role of the cytoskeleton in stimulation of Na+ channels in A6 cells by changes in osmolality

 Permeable supports with A6 cell monolayers were mounted in an Ussing chamber and bilaterally bathed with Ringer solution at room temperature. Short-circuit current (I _sc) was recorded continuously, and noise analysis revealed microscopic channel current characteristics. Our investigation focuses on the stimulation of apical Na⁺ entry caused by exposing the serosal surface of the A6 cell monolayers to hyposmotic Ringer solution. To evaluate the possible role of the cytoskeleton in the regulation of Na⁺ channels in response to a change in osmolality we used four different experimental approaches. In the control group, which were not exposed to any cytoskeleton-influencing drugs, there was a 1.5-fold increase in I _sc and in the number of open Na⁺ channels after osmotic stimulation. For the second group cytochalasin D (0.1 μg/ml) was present on the serosal side during the experiments. Neither I _sc nor the number of open Na⁺ channels increased after osmotic stimulation. In the third group colchicine (0.2 mM) or nocodazole (20 μM) was present on the serosal side, which resulted in 1.8-fold and 1.5-fold increases in I _sc as well as 3-fold and 2-fold increases in the number of Na⁺ channels, respectively. In the fourth experimental group erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride (EHNA, 0.5 mM), a dynein inhibitor, was present on the serosal side. In this group I _sc decreased to about 0.4 μA/cm², and subsequent application of amiloride abolished I _sc completely. Under hyposmolar conditions EHNA abolished entirely the sensitivity of I _sc to the osmotic challenge. Because of the EHNA-induced down-regulation of I _sc, the density of apical Na⁺ channels in this experimental group could not be determined. These results show that the cytoskeleton is dominantly involved in osmotic channel regulation at the apical membrane, and that actin filaments, microtubules and molecular motors are involved in the recruitment of additional Na⁺ channels. Received: 21 July 1997 / Received after revision: 4 December 1997 / Accepted: 16 December 1997     

Voltage-gated Na+ current availability after step- and spike-shaped conditioning depolarizations of retinal ganglion cells

 We used two conditioning voltage protocols to assess inactivation of voltage-gated Na⁺ current in retinal ganglion cells. The first protocol tested the possibility, raised by published activation and steady-state inactivation curves, that Na⁺ ions carry a ”window” current in these cells. The second protocol was used, because these cells spike repetitively in situ, to measure the Na⁺ current available for activation following spikes. Na⁺ current activated at test potentials more positive than –65 mV. At test potentials more positive than –55 mV, Na⁺ current peaked and then declined along a time course that could be fit by the sum of a large, rapidly decaying component, a small, slowly decaying component and a non-decaying component. Both step- and spike-shaped conditioning depolarizations reduced the amount of current available for subsequent activation, sparing the non-decaying ”persistent” component. Most of the Na⁺ current recovered from this inactivation along a rapid exponential time course (τ=3 ms). The remaining recovery was complete within at least 4 s (at –70 mV). Our use of step depolarizations has identified a current component not anticipated from previous measurements of steady-state inactivation in retinal ganglion cells. Our use of spike-shaped depolarizations shows that Na⁺ current density at 1 ms after a single spike is roughly 25% of that activated by the conditioning spike, and that recovery from inactivation is 50–90% complete within 10 ms thereafter. Na⁺ current amplitude declines during spikes repeated at relatively low frequencies, consistent with a slow component of full recovery from inactivation. Received: 9 October 1997 / Received after revision: 30 March 1998 / Accepted: 15 April 1998     

Effect of dehydration on apical Na+-H+ exchange activity and Na+-dependent sugar transport in brush-border membrane vesicles isolated from chick intestine

 The current work examines the effect of 4 days of water deprivation on Na⁺-H⁺ exchange and Na⁺-sugar cotransport systems in brush-border membrane vesicles isolated from either the jejunum, ileum or the colon of the chick. Apical Na⁺-H⁺ exchange activity was evaluated by measuring the pH-gradient-dependent Na⁺ uptake. The contribution of the Na⁺-H⁺ exchangers NHE2 and NHE3 to total Na⁺-H⁺ exchange activity was evaluated from their sensitivity to the amiloride-related drug HOE694. Dehydration increased plasma aldosterone levels from 12 to 70 pg/ml and also the activities of both Na⁺-H⁺ exchange and Na⁺-dependent sugar transport in the three intestinal regions tested. Na⁺-independent sugar transport was not modified by 4 days of water deprivation. In the ileum and colon the increase in Na⁺-H⁺ exchange activity was due to an increase in NHE2 activity, whereas in the jejunum it was due to an increase in both NHE2 and NHE3. Since we have previously reported that chronic Na⁺ depletion increases plasma aldosterone levels and NHE2 activity in ileum and colon, decreased small and large intestine Na⁺-sugar cotransport activity and had no effect on jejunal apical Na⁺-H⁺ exchange activity, it can be concluded that: (1) aldosterone does not regulate intestinal Na⁺-dependent sugar transport, and (2) the regulation of jejunal Na⁺-H⁺ exchange activity differs from that of either the ileum or the colon. Received: 31 October 1997 / Received after revision: 17 December 1997 / Accepted: 8 January 1998     

Effects of reduced electrochemical Na+ gradient on contractility in skeletal muscle: role of the Na+-K+ pump

 Continued excitation of skeletal muscle may induce a combination of a low extracellular Na⁺ concentration ([Na⁺]_o) and a high extracellular K⁺ concentration ([K⁺]_o) in the T-tubular lumen, which may contribute to fatigue. Here, we examine the role of the Na⁺-K⁺ pump in the maintenance of contractility in isolated rat soleus muscles when the Na⁺, K⁺ gradients have been altered. When [Na⁺]_o is lowered to 25 mM by substituting Na⁺ with choline, tetanic force is decreased to 30% of the control level after 60 min. Subsequent stimulation of the Na⁺-K⁺ pump with insulin or catecholamines induces a decrease in [Na⁺]_i and hyperpolarization. This is associated with a force recovery to 80–90% of the control level which can be abolished by ouabain. This force recovery depends on hyperpolarization and is correlated to the decrease in [Na⁺]_i (r = 0.93; P<0.001). The inhibitory effect of a low [Na⁺]_o on force development is considerably potentiated by increasing [K⁺]_o. Again, stimulation of the Na⁺-K⁺ pump leads to rapid force recovery. The Na⁺-K⁺ pump has a large potential for rapid compensation of the excitation-induced rundown of Na⁺, K⁺ gradients and contributes, via its electrogenic effect, to the membrane potential. We conclude that these actions of the Na⁺-K⁺ pump are essential for the maintenance of excitability and contractile force. Received: 19 December 1996 / Received after revision: 25 March 1997 / Accepted: 2 April 1997     

Secretory apical Cl– channels in A6 cells: possible control by cell Ca2+ and cAMP

Distal kidney cells (A6) from Xenopus laevis were cultured to confluency on porous supports. Tissues were mounted in Ussing-type chambers to measure short-circuit current (I _sc), transepithelial conductance and capacitance, and to analyse the fluctuation in I _sc. In the absence of apical NaCl, but with normal basolateral NaCl Ringer’s solution, extracellular addition of ATP, oxytocin, a membrane-permeant cAMP derivative, and forskolin produced a transient increase of the electrical parameters. Noise analysis revealed a spontaneous Lorentzian component. All responses depend strictly on the presence of basolateral Cl^– and are caused by the activation of an apical (CFTR type) Cl^– permeability. Repetitive treatment with ATP (or oxytocin) resulted in refractoriness. ATP and oxytocin acted antagonistically, whereas cAMP and ATP had additive effects. Incubation with the vesicular Ca²⁺ pump inhibitor thapsigargin or application of the Ca²⁺ channel blocker nifedipine elicited finite but variable Cl^– channel activity. After treatment with nifedipine or thapsigargin, the response to oxytocin was severely impaired. We speculate that not only cAMP but also cell Ca²⁺ plays a crucial role in the activation of CFTR in A6. Ca²⁺ may be multifunctional but the rise in capacitance (apical area) observed with all stimulants strongly suggests its involvement in, and contribution to, exocytosis in the process of the CFTR-mediated transcellular Cl^– movements. Received: 30 November 1998 / Received after revision: 23 February 1999 / Accepted: 12 March 1999     

Vanadyl ions stimulate K+ uptake into isolated perfused rat liver via the Na+/K+-pump by a tyrosine kinase-dependent mechanism

 Vanadium salts mimic most metabolic effects of insulin in vitro. We report here that vanadyl sulfate (VOSO₄) and sodium vanadate (NaVO₃) stimulate net K⁺ uptake in isolated perfused rat liver. Stimulation was evident at low concentrations of vanadyl ions (range 1–20 μM) and occurred within minutes following the addition of VOSO₄. By comparison with VOSO₄, insulin had less of a stimulatory effect on K⁺ uptake. Ouabain prevented the activating effect of VOSO₄ on K⁺ uptake. Following a VOSO₄ challenge, measured intracellular Na⁺ concentration ([Na⁺]_i) fell (control, 17.1 ± 1.2; VOSO₄-treated, 13.0 ± 1.1 mmol·g^–1 wet weight, P = 0.027). The results indicate that active K⁺ uptake via the Na⁺/K⁺-ATPase was stimulated by vanadyl ions. An indirect mechanism due to changes in [Na⁺]_i can be excluded. The tyrosine kinase inhibitor genistein was found to inhibit stimulation of K⁺ by vanadyl and vanadate ions which are known inhibitors of phosphotyrosine phosphatases. We conclude that stimulation of active K⁺ influx involves a tyrosine kinase. Possible mechanisms include phosphorylation at tyrosine residues and direct activation of the Na⁺/K⁺-ATPase, or phosphorylation of other proteins that regulate the activity or number of pumps in the cells. Received: 8 July 1997 / Received after revision: 3 November 1997 / Accepted: 11 November 1997     

Voltage-dependent gating of veratridine-modified RIIA Na+ channel α subunit expressed heterologously in CHO cells

The voltage-dependent kinetics of veratridine-modified RIIA Na⁺ channel α subunit expressed heterologously in CHO cells were studied using the whole-cell patch-clamp technique. The activation and deactivation kinetics are well described by double exponential functions but poorly by a monoexponential function. Unlike the slow component, the fast time constant and associated amplitude factor depended steeply on the potential. The steady-state activation of veratridine-modified channels is described by a Boltzmann function with a V _1/2 of –131.9 mV and a slope of 9.41 mV. A two-state model is proposed for the fast component that explains the kinetics of veratridine’s mechanism of action. Received: 11 December 1998 / Received after revision: 23 March 1999 / Accepted: 7 April 1999     

A common local anesthetic receptor for benzocaine and etidocaine in voltage-gated μ1 Na+ channels

 According to Hille’s modulated receptor hypothesis, benzocaine shares a common receptor with all other local anesthetics (LAs) in the voltage-gated Na⁺ channel. We tested this single receptor hypothesis using mutant muscle Na⁺ channels of μ1-I1575A, F1579A, and N1584A transiently expressed in Hek-293t cells. Both benzocaine and etidocaine are more effective at blocking μ1-N1584A current than the wild-type current, while they are less potent at blocking μ1-F1579A current. Such concurrent changes of both benzocaine and etidocaine potency towards F1579A and N1584A mutants suggest that they share a common LA receptor. Consistent with results found in studies of native Na⁺ channels, permanently charged QX-314 at 1 mM is not effective at blocking wild-type, F1579A, and N1584A current via external application. In contrast, QX-314 is relatively potent at blocking I1575A current when applied externally. This increased potency of external QX-314 against the μ1-I1575A mutant has been reported previously in a study of the brain counterpart. Mutant I1575A also appears to be highly sensitive to the external divalent cation Cd²⁺, probably because of the presence of cysteine residues near the μ1-I1575 position in the IV-S6 segment. To our surprise, neutral benzocaine becomes more effective at blocking μ1-I1575A current than the wild-type current, whereas the opposite is found for etidocaine. We hypothesize that an increase in accessibility of external QX-314 to the μ1-I1575A mutant is accompanied by a reduction of binding towards the charged amine component. Received: 27 May 1997 / Received after revision: 29 August 1997 / Accepted: 5 September 1997     

A Na+-activated K+ current (I K,Na) is present in guinea-pig but not rat ventricular myocytes

 The effects of removing extracellular Ca²⁺ and Mg²⁺ on the membrane potential, membrane current and intracellular Na⁺ activity (a ⁱ _Na) were investigated in guinea-pig and rat ventricular myocytes. Membrane potential was recorded with a patch pipette and whole-cell membrane currents using a single-electrode voltage clamp. Both guinea-pig and rat cells depolarize when the bathing Ca²⁺ and Mg²⁺ are removed and the steady-state a ⁱ _Na increases rapidly from a resting value of 6.4± 0.6 mM to 33±3.8 mM in guinea-pig (n=9) and from 8.9±0.8 mM to 29.3±3.0 mM (n=5) in rat ventricular myocytes. Guinea-pig myocytes partially repolarized when, in addition to removal of the bathing Ca²⁺ and Mg²⁺, K⁺ was also removed, however rat cells remained depolarized. A large diltiazem-sensitive inward current was recorded in guinea-pig and rat myocytes, voltage-clamped at –20 mV, when the bathing divalent cations were removed. When the bathing K⁺ was removed after Ca²⁺ and Mg²⁺ depletion, a large outward K⁺ current developed in guinea-pig, but not in rat myocytes. This current had a reversal potential of –80±0.7 mV and was not inhibited by high Mg²⁺ or glybenclamide indicating that it is not due to activation of non-selective cation or adenosine triphosphate (ATP)-sensitive K channels. The current was not activated when Li⁺ replaced the bathing Na⁺ and was blocked by R-56865, suggesting that it was due to the activation of K_Na channels. Received: 15 October 1998 / Received after revision: 22 January 1999 / Accepted: 5 February 1999     

Activation of Ca2+-sensitive Cl– current by reverse mode Na+/Ca2+ exchange in rabbit ventricular myocytes

 We investigated how Ca²⁺-sensitive transient outward current, I _to(Ca), is activated in rabbit ventricular myocytes in the presence of intracellular Na⁺ (Na⁺ _i) using the whole-cell patch-clamp technique at 36°C. In cells dialysed with Na⁺-free solutions,the application of nicardipine (5 μM) to block L-type Ca²⁺ current (I _Ca) completely inhibited I _to(Ca). In cells dialysed with a [Na⁺]_i≥5 mM, however, I _to(Ca) could be observed after blockade of I _Ca, indicating the activity of an I _Ca-independent component. The amplitude of I _Ca-independent I _to(Ca) increased with voltage in a [Na⁺]_i-dependent manner. The block of Ca²⁺ release from the sarcoplasmic reticulum by caffeine, ryanodine or thapsigargin blocked I _Ca-independent I _to(Ca). In Ca²⁺-free bath solution I _to(Ca) was completely abolished. The application of 2 mM Ni²⁺ or the newly synthesized compound KBR7943, a selective blocker of the reverse mode of Na⁺/Ca²⁺ exchange, or perfusion with pipette solution containing XIP (10 μM), a selective blocker of the exchanger, blocked I _Ca-independent I _to(Ca). From these results we conclude that, in the presence of Na⁺ _i, I _to(Ca) can be activated via Ca²⁺-induced Ca²⁺ release triggered by Na⁺/Ca²⁺ exchange operating in the reverse mode after blockade of I _Ca. Received: 20 January 1998 / Received after revision: 6 July 1998 / Accepted: 25 July 1998     

Characterization of the Na+/H+ exchanger in human choriocarcinoma (BeWo) cells

 We examined the expression and activity of the Na⁺/H⁺ exchanger in the human choriocarcinoma BeWo cell line. When treated with methotrexate, these cells differentiated from cytotrophoblast-like cells to enlarged multinucleate syncytiotrophoblast-like cells. There was no change in the apparent K _m for Na⁺ between undifferentiated and differentiated cells. However, differentiated cells could transport more than five times the proton flux of undifferentiated cells. There was no difference in the Hill coefficient between undifferentiated and differentiated cells. However, the maximal flux (J _max) for undifferentiated cells was higher than that for differentiated cells. Inhibition of Na⁺/H⁺ exchange activity by an amiloride analog and Hoe694 revealed a sensitive and a resistant component in both differentiated and undifferentiated cells. Northern blot analysis and immunocytochemistry suggested that the sensitive component was due to the NHE1 isoform of the protein while the resistant component was due to the NHE3 isoform. The NHE1 isoform was localized to the brush border membrane of BeWo cells and Western blot analysis showed that the NHE1 protein was more abundant in brush border membranes from differentiated BeWo cells compared to undifferentiated cells. The results show that BeWo cells contain the NHE1 and NHE3 isoforms of the Na⁺/H⁺ exchanger and that the NHE1 isoform is primarily localized to the brush border membrane. Received: 25 July 1996 / Received after revision: 25 Novembber 1996 / Accepted: 3 December 1996     

Digitalis-like compounds and Na+, K+-ATPase activity in bovine lens

 Digitalis-like compounds in bovine lens capsule, cortex and nucleus were determined quantitatively, following extraction, by their ability to inhibit [³H]ouabain binding to red blood cells. These compounds were found to be highly concentrated in the epithelium capsule and were significantly diminished in the cortex and nucleus. Na⁺, K⁺-ATPase density in the different regions was determined by [³H]ouabain binding to membranes and by autoradiography of lens slices. The highest concentration of [³H]ouabain-binding sites was observed to occur in membranes prepared from the epithelial cells of the capsule, and was almost 100- and 200-fold higher than the concentrations observed in membranes prepared from fiber cells of the cortex and nucleus, respectively. In the autoradiography studies, strong labeling of [³H]ouabain appeared in the epithelial cell zone, and only weak specific labeling appeared in the lens cortex and nucleus. Almost all (99%) of the Na⁺,K⁺-ATPase specific activity was found to be in the capsule epithelium and only 0.5% was measured in the cortex and no activity was detected in the nucleus. These results indicate that the digitalis-like compounds and Na⁺, K⁺-ATPase are concentrated in the lens capsule epithelium and are present only at low levels in the cortex and nucleus, thus implying that the lens capsular epithelial layer is the major region of the lens responsible for the homeostasis of ions and water in this tissue. Received: 20 September 1996 / Received after revision and accepted: 17 October 1996     

Insulin effects on ouabain binding in A6 renal cells

 The effects of insulin on the Na⁺-K⁺-ATPase pump of the basolateral membrane of tight epithelia were evaluated by measuring transepithelial transport and [³H]ouabain binding in cultured A6 kidney cells. [³H]Ouabain binding in epithelia incubated in either K⁺-containing or K⁺-free solutions was measured. Insulin induced increases in transepithelial sodium transport, as measured by the short-circuit current (I _sc), and in the initial rate of [³H]ouabain binding determined when the preparation was bathed in K⁺-containing solutions. However, when initial [³H]ouabain binding in tissues incubated in K⁺-free solutions was measured the stimulation of the initial rate of [³H]ouabain binding caused by insulin was markedly reduced. Incubating the apical side of the epithelium with either amiloride or Na⁺-free solutions also reduced or abolished the increase in the initial rate of [³H]ouabain binding caused by insulin. Equilibrium binding measurements showed that insulin did not increase the maximum number of [³H]ouabain-binding sites in tissues incubated with either normal K⁺ or K⁺-free solutions. These results indicate that the increase in the initial rate of [³H]ouabain binding under transporting conditions is due to an effect on the binding kinetics of ouabain, probably related to an increased rate of Na⁺ entry, rather than to an increase in the number of Na⁺-K⁺-ATPases in the basolateral membrane. Cycloheximide inhibited both the increase in I _sc and the increase in the initial rate of [³H]ouabain binding caused by insulin in epithelia incubated in K⁺-containing solutions. However, cycloheximide was without effect on the initial rate of [³H]ouabain binding in insulin-treated tissues incubated in K⁺-free solution. This finding suggests that the cycloheximide-sensitive step of the action of insulin is related to Na⁺ delivery to the pump. Received: 2 October 1995 / Received after revision: 6 January 1997 / Accepted: 13 January 1997     

Calcium channel current facilitation in porcine adrenal chromaffin cells

 The mechanisms of depolarizing-prepulse-induced facilitation of Ca²⁺ channel current were investigated in a study of porcine chromaffin cells. The Ba²⁺ current evoked by a pulse to 0 mV was increased by a strong depolarizing prepulse (conditioning pulse), termed ”facilitation”. This facilitation increased with an increase in either the duration or the voltage of the conditioning pulse, and decreased with an increase in the interpulse interval. For example, the Ba²⁺ current was increased to 1.14 times the control (facilitation ratio) by a 150-ms conditioning pulse to +100 mV followed by a 10-ms interpulse interval. Forskolin, 8-bromo-adenosine 3′,5′-cyclic monophosphate (8-bromo-cAMP) and Rp-adenosine 3′,5′-cyclic monophosphothioate (Rp-cAMPS) did not affect the facilitation of the Ba²⁺ current, suggesting that a cAMP-dependent mechanism is not involved. Intracellular guanosine 5′-O-(3-thiotriphosphate) (GTPγS) decreased the Ba²⁺ current to 0.59 times the control and GDPβS increased it to 1.19. However, neither GTPγS nor guanosine 5′-O-(2-thiodiphosphate) (GDPβS) changed the amplitude of the Ba²⁺ current that was facilitated by the conditioning pulse. Thus, GTPγS increased the facilitation ratio to 2.05 and GDPβS decreased it to 1.05. Furthermore, the facilitation of the Ba²⁺ current was abolished by ω-conotoxin GVIA but not by either ω-agatoxin IVA or nifedipine. These results suggest that, in porcine chromaffin cells, there is a ω-conotoxin GVIA-sensitive N-type Ca²⁺ channel that is under the inhibitory control of a G protein, which can be relieved by a conditioning pulse. Received: 25 September 1997 / Received after revision: 14 November 1997 / Accepted: 16 December 1997