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     

Expression and function of Na+/Ca2+ exchangers 1 and 3 in human macrophages and monocytes

The Na⁺/Ca²⁺ exchanger (NCX) is a membrane transporter that can switch Na⁺ and Ca²⁺ in either direction to maintain the homeostasis of intracellular Ca²⁺. Three isoforms (NCX1, NCX2, and NCX3) have been characterized in excitable cells, e.g. neurons and muscle cells. We examined the expression of these NCX isoforms in primary human lung macrophages (HLM) and blood monocytes. NCX1 and NCX3, but not NCX2, are expressed in HLM and monocytes at both mRNA and protein levels. Na⁺‐free medium induced a significant increase in intracellular calcium concentration ([Ca²⁺]_i) in both cell types. This response was completely abolished by the NCX inhibitor 5‐(N‐4‐chlorobenzyl)‐20,40‐dimethylbenzamil (CB‐DMB). Moreover, inhibition of NCX activity during Ca²⁺‐signaling induced by histamine caused a delay in restoring baseline [Ca²⁺]_i. Na⁺‐free medium induced TNF‐α expression and release in HLM comparable to that caused by LPS. TNF‐α release induced by Na⁺‐free medium was blocked by CB‐DMB and greatly reduced by RNAi‐mediated knockdown of NCX1. These results indicate that human macrophages and monocytes express NCX1 and NCX3 that operate in a bidirectional manner to restore [Ca²⁺]_i, to generate Ca²⁺‐signals, and to induce TNF‐α production. Therefore, NCX may contribute to regulate Ca²⁺ homeostasis and proinflammatory functions in human macrophages and monocytes.     

Calcium fluxes in rat thyroid FRTL-5 cells. Evidence for a functional Na+/Ca2+ exchange mechanism

Törnquist , K. 1992. Calcium fluxes in rat thyroid FRTL-5 cells. Evidence for a functional Na⁺/Ca²⁺ exchange mechanism. Acta Physzol Scand 144 , 341–348. Received 28 April 1 991 , accepted 30 October 1991. ISSN 0001–6772. Endocrine Research Laboratory, University of Helsinki, Minerva Foundation Institute for Medical Research, Helsinki, Finland. The effect of extracellular Na⁺ on cytosolic free Ca²⁺ and on influx and efflux of Ca²⁺ was investigated in FRTL-5 thyroid cells. Stimulating the cells with the purinergic agonist ATP induced a rapid efflux of ⁴⁵Ca²⁺ from cells loaded with ^4aCa²⁺. Replacement of extracellular Na⁺ with choline⁺, significantly decreased the adenosine triphosphate-induced efflux of ⁴⁵Ca²⁺. Furthermore, adenosine triphosphate-induced uptake of ⁴⁵Ca²⁺ was increased when extracellular Na⁺ was replaced with choline⁺, compared with the uptake seen in Na⁺ buffer. Replacing extracellular Na⁺ with choline⁺, increased resting levels of cytosolic free Ca²⁺ from 50 ± 2 nM (mean ± SE) to 81 ± 3 nM (P < 0.05) in Fura 2 loaded cells. In cells preincubated with 1 mM ouabain for 30 min, resting cytosolic free Ca²⁺ increased to 73 ± 3 nM (P < 0.05). In a Na⁺ buffer, the adenosine triphosphate-induced transient increase in cytosolic free Ca²⁺ was 872 ± 59 nM, compared with 1070 ±63 nM in choline' buffer (P < 0.05). The plateau level of cytosolic free Ca²⁺ in response to adenosine triphosphate was 130±16 nM in Na⁺ buffer, compared with 209±9 nM in choline⁺ buffer (P < 0.05). Readdition of Na⁺ to the plateau phase decreased cytosolic free Ca⁺² to 152 ± 5 nM. Stimulating the cells with 10 μM of the Na^+-selective monovalent ionophore monensin increased cytosolic free Ca²⁺ from 53 ± 9 nM to 12416 nM (P < 0.05). This increase in cytosolic free Ca²⁺ was dependent on both extracellular Na⁺ and extracellular Ca²⁺ If cells were first stimulated with monensin, and then with adenosine triphosphate, the transient increase in cytosolic free Ca²⁺ was 1027 ± 24 nM (P < 0. 05 , compared with control cells). The results thus indicate, that FRTL-5 cells have a functional Na⁺/Ca²⁺ exchange mechanism and that this mechanism is of importance in restoring adenosine triphosphate-induced transient increase in cytosolic free Ca²⁺ to resting cytosolic free Ca²⁺ levels.     

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     

S3226, a novel inhibitor of Na+/H+ exchanger subtype 3 in various cell types

 Inhibition of Na⁺/H⁺ exchange (NHE) subtypes has been investigated in a study of the mouse fibroblast L cell line (LAP1) transfected with human (h) NHE1, rabbit (rb) NHE2, rat (rt) or human (h) NHE3 as well as an opossum kidney cell line (OK) and porcine renal brush-border membrane vesicles (BBMV). S3226 {3-[2-(3-guanidino-2-methyl-3-oxo-propenyl)-5-methyl-phenyl]-N-isopropylidene-2-methyl-acrylamide dihydro-chloride} was the most potent and specific NHE3 inhibitor with an IC₅₀ value of 0.02 μmol/l for the human isoform, whereas its IC₅₀ value for hNHE1 and rbNHE2 was 3.6 and @80 μmol/l, respectively. In contrast, amiloride is a weak NHE3 inhibitor (IC₅₀>100 μmol/l) with a higher affinity to hNHE1 and rbNHE2. Cariporide (4-isopropyl-3-methylsulphonyl-benzoyl-guanidine methane-sulphonate), which has an IC₅₀ for NHE3 of approximately 1 mmol/l, is a highly selective NHE1 inhibitor (0.08 μmol/l). Therefore, S3226 is a novel tool with which to investigate the physiological and pathophysiological roles of NHE3 in animal models. Received: 14 May 1998 / Received after revision: 29 June 1998 / Accepted: 2 July 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     

Selective inhibition of the Na+/H+ exchanger type 3 activates CO2/H+-sensitive medullary neurones

Hypercapnia as well as lowered intracellular pH (pH_i) increase the bioelectric activity of CO₂/H⁺-sensitive neurones (VLNcs) of the ventrolateral medulla oblongata. Here we describe that immunoreactive Na⁺/H⁺ exchanger (NHE3) is present in ventrolateral neurones from medullary organotypic cultures (obex level). To test whether VLNcs can be acidified and thereby activated by inhibition of NHE3, we used the novel high-affinity NHE3-inhibitors S1611 and S3226. Both drugs raised the firing rates of VLNcs to at least 150% of the control values, and depolarized membrane potential by up to 15 mV at concentrations (0.5–1 μmol/l) suitable for selective inhibition of NHE3. The changes in bioelectric activity strongly resembled the responses to hypercapnia (PCO₂: 60–100 mmHg). In BCECF-AM-loaded cultures a subfraction of ventrolateral VLNcs was found to be intracellularly acidified by 0.05–0.1 pH units following treatment with S1611; the time course of this acidification was similar to that evoked by hypercapnia. All drug effects were sustained and readily reversible upon washing. Non-CO₂/H⁺-responsive medullary neurones as well as hippocampal CA3 neurones were unaffected by up to 20 μmol/l S1611. It is concluded that the selective inhibition of NHE3 acidifies and activates CO₂/H⁺-sensitive neurones within the ventrolateral medulla oblongata. Received: 12 February 1999 / Received after revision and accepted: 15 April 1999     

Kinetic components of the gating currents of human cardiac L-type Ca2+ channels

 It has been reported previously that the β subunit increases both the ionic current and the gating charge movement of the human cardiac L-type Ca²⁺ channel α₁ subunit, and that steady-state measurements reveal the presence of two distinct components of the charge movement [Josephson IR, Varadi G (1996) Biophys J 70:1285–1293]. The present work identifies and characterizes the kinetic properties of the components of the human cardiac L-type Ca channel gating currents (I _g), and determines the relationship of these components to the activation of the Ca channel ionic current (I _Ca). Cloned human cardiac L-type α₁+α₂+β₃ subunits were transiently expressed in HEK293 cells and calcium channel gating currents were recorded following the addition of 5 mM Co²⁺. The steady-state charge integrals of the gating currents (Q _ON-V _m) were fit by a sum of two Boltzmann components: Q _ON1, which ranged over more negative potentials, and Q _ON2, which ranged over more positive potentials. The kinetic components of the ON and OFF gating currents were identified using bi-exponential curve fitting. Reconstruction of the two kinetic components of charge (Q _ONfast and Q _ONslow) yielded distributions that were similar in their voltage dependence and relative proportion to those measured directly by steady-state integration of Q _ON1 and Q _ON2. Changes in the initial conditions were found to affect Q _ON1 and Q _ON2 differently. The time constants of the ON gating current decays were similar to those of the activation of I _Ca. The results suggest that: (1) the activation of the human cardiac L-type Ca channel involves the movements of at least two, functionally distinct gating structures; (2) a fast charge movement (≈1/4 of the total charge; Q _ON1 or Q _ONfast) precedes a slower charge movement (≈3/4 of the total charge; Q _ON2 or Q _ONslow); and (3) channel opening is associated with the conformational change(s) producing Q _ONslow. Received: 7 June 1996 / Received after revision: 24 September 1996 / Accepted: 1 October 1996     

Ruthenium red reduces the Ca2+ sensitivity of Ca2+ uptake into cardiac sarcoplasmic reticulum

 Ruthenium red inhibits mitochondrial Ca²⁺ uptake and is widely used as an inhibitor of ryanodine-sensitive Ca²⁺ channels that function to release Ca²⁺ from the sarcoplasmic reticulum (SR) of muscle cells. It also has effects on other Ca²⁺ channels and ion transporters. To study the effects of ruthenium red on Ca²⁺ transport into the SR of cardiac muscle cells, fluorescence measurements of Ca²⁺ uptake into cardiac SR vesicles were made. Ruthenium red significantly decreased the Ca²⁺ sensitivity of SR uptake in a dose-dependent manner at concentrations ranging from 5 μM to 20 μM. There were no significant effects of ruthenium red on the maximum velocity or the Hill coefficient of SR Ca²⁺ uptake. Received: 14 January 1998 / Received after revision: 12 March 1998 / Accepted: 16 March 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     

Caffeine stimulates the reverse mode of Na+/Ca2+ exchanger in ferret ventricular muscle

This study investigated the effect of caffeine on the sarcolemmal mechanisms involved in intracellular calcium control. Ferret cardiac preparations were treated with ryanodine and thapsigargin in order to eliminate the sarcoplasmic reticulum (SR) function. This treatment abolished caffeine contracture irreversibly in normal solution. The perfusion with K‐free medium that blocked the Na⁺–K⁺ pump resulted in a recovery of slow relaxing caffeine contractures similar to Na‐free contractures. The amplitude of caffeine contractures was dependent on the bathing [caffeine]o and [Ca²⁺]_o. Divalent cations Ni²⁺ and Cd²⁺, which have an inhibitory effect on the Na⁺/Ca²⁺ exchanger, produced dose‐dependent inhibition of caffeine responses with apparent Ki of 780 ± 19 and 132 ± 5 μM , respectively. Caffeine also caused dose‐dependent inhibition of Na‐free contractures (Ki=4.62 ± 1.5 mM ), and the reduction or removal of [Na⁺]_o exerted an inhibitory effect on caffeine contractures (Ki=73.5 ± 17.12 mM ). These experiments indicate that the increase in resting tension following exposure to caffeine was mediated by Na⁺/Ca²⁺ exchanger, which represents an additional element of complexity in caffeine action on cardiac muscle.     

Ca2+ entry through cardiac L-type Ca2+ channels modulates β-adrenergic stimulation in mouse ventricular myocytes

 β-adrenergic receptor (β-AR) stimulation increases cardiac L-type Ca²⁺ channel (CaCh) currents via cAMP-dependent phosphorylation. We report here that the affinity and maximum response of CaCh to isoproterenol (Iso), in mouse ventricular myocytes were significantly higher when Ba²⁺ was used as the charge carrier (I _Ba) instead of Ca²⁺ (I _Ca). The EC₅₀ and maximum increase of peak currents were 43.7 ± 7.9 nM and 1.8 ± 0.1-fold for I _Ca and 23.3 ± 4.7 nM and 2.4 ± 0.1-fold for I _Ba. When cells were dialyzed with the faster Ca²⁺ chelator, BAPTA, both sensitivity and maximum response of I _Ca to Iso were significantly augmented compared to cells with EGTA (EC₅₀ of 23.1 ± 5.2 nM and maximal increase of 2.2 ± 0.1-fold). Response of I _Ca to forskolin was also significantly increased when cells were dialyzed with BAPTA or when currents were measured in Ba²⁺. In contrast, depletion of the sarcoplasmic reticulum (SR) Ca²⁺ stores by ryanodine did not alter sensitivity of I _Ca to Iso or forskolin. These results suggest that the Ca²⁺ entering through CaCh regulates cAMP-dependent phosphorylation, and such negative feedback may play a significant role in cellular Ca²⁺ homeostasis and contraction in cardiac cells during β-AR stimulation. Received: 10 December 1997 / Received after revision: 19 January 1998 / Accepted: 21 January 1998     

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     

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     

[Ca2+]i elevation evoked by Ca2+ readdition to the medium after agonist-induced Ca2+ release can involve both IP 3-, and ryanodine-sensitive Ca2+ release

 Sustained Ca²⁺ elevation (”Ca²⁺ response”), caused by subsequent readdition of Ca²⁺ to the medium after application of adenosine 5’-triphosphate (ATP, 15 μM) in a Ca²⁺-free medium, was studied using single bovine aortic endothelial (BAE) cells. In cells in which the resting intracellular Ca²⁺ concentration ([Ca²⁺]_i) was between about 50 and 110 nM, a massive Ca²⁺ response occurred and consisted of phasic and sustained components, whereas cells with a resting [Ca²⁺]_i of over 110 nM displayed small plateau-like Ca²⁺ responses. An increase of internal store depletion resulted in loss of the phasic component. When the store was partly depleted, the dependence of the Ca²⁺ response amplitude on resting [Ca²⁺]_i was biphasic over the range of 50 to 110 nM. The greatest degree of store depletion was associated with small monophasic Ca²⁺ responses, the amplitudes of which were almost constant and in the same range as resting [Ca²⁺]_i. Ni²⁺, known to partly block Ca²⁺ entry, caused no change in the half-decay time of [Ca²⁺]_i down to the level of the sustained phase [57 ± 4 s in control and 54 ± 3 s (n = 13) in the presence of 10 mM Ni²⁺] when added at the peak of the phasic component of the Ca²⁺ response. However, it lowered the sustained phase of the Ca²⁺ response by 42%. When applied at the start of the readdition of Ca²⁺, Ni²⁺ blocked the phasic component of the Ca²⁺ response, there being a threefold decrease in the initial rate of [Ca²⁺]_i rise. In cells with a resting [Ca²⁺]_i of 75–80 nM, pre-treatment with ryanodine (10 μM) did not affect the peak amplitude of the Ca²⁺ response, but it did increase the level of the sustained component. In some cells, ryanodine caused an oscillatory Ca²⁺ response. In conclusion, partial depletion of the inositol 1,4,5-trisphosphate-(IP ₃-) sensitive store by a submaximal concentration of agonist (in Ca²⁺-free medium) was followed, on readdition of Ca²⁺, by Ca²⁺ entry, which appeared to trigger IP ₃-sensitive Ca²⁺ release (IICR) which, in turn, initiated Ca²⁺-sensitive Ca²⁺ release (CICR), thus resulting in a massive elevation of [Ca²⁺]_i. Received: 3 July 1996 / Received after revision and accepted: 9 September 1996     

Involvement of Ca2+-induced Ca2+ release in the biphasic Ca2+ response evoked by readdition of Ca2+ to the medium after UTP-induced store depletion in A431 cells

 We have recently shown that the Ca²⁺ response in endothelial cells evoked by readdition of Ca²⁺ to the medium after store depletion caused by a submaximal concentration of agonist can involve Ca²⁺ release from Ca²⁺ stores sensitive to both inositol 1,4,5-trisphosphate and ryanodine. The present experiments were performed to determine whether this mechanism might also exist in other types of cell. For this purpose, we used the human carcinoma cell line A431, which has a varied resting [Ca²⁺]_i. We found that the amplitude of the Ca²⁺ response evoked by Ca²⁺ readdition did not correlate with the amplitude of the preceding UTP-evoked Ca²⁺ release, but did positively correlate with the initial [Ca²⁺]_i. An inspection of the two patterns of response seen in this study (the large biphasic and small plateau-shaped Ca²⁺ responses) revealed that there is an accelerating rise in [Ca²⁺]_i during the biphasic response. Application of ryanodine during the plateau-shaped Ca²⁺ response reversibly transformed it into the biphasic type. Unlike ryanodine, caffeine did not itself evoke Ca²⁺ release, but it caused a further [Ca²⁺]_i rise when [Ca²⁺]_i had already been elevated by thapsigargin. These data suggest that in A431 cells, as in endothelial cells, the readdition of Ca²⁺ after agonist-evoked store depletion can evoke Ca²⁺-induced Ca²⁺ release. This indicates that Ca²⁺ entry may be overestimated by this widely used protocol. Received: 28 July 1997 / Received after revision: 25 November 1997 / Accepted: 26 November 1997     

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