一种新的钠-钙交换电流记录方法及阿米洛利的作用

目的：研究胞外不同Ca²⁺浓度对豚鼠心室肌细胞钠-钙交换电流(Na⁺-Ca²⁺ exchange current, INa-Ca)的影响和阿米洛利(amiloride)对该电流的作用。方法：建立缺血再灌时胞内Na⁺超载的细胞模型,用膜片钳全细胞技术,记录I_Na-Ca的电流-电压关系曲线。结果：阿米洛利10^-5,3×10^-5和10^-4 mol.L^-1,在+50 mV时,对I_Na-Ca的抑制率分别是15.4%,22.6%和40.9%;在-80 mV时抑制率分别是5.6%,14.6%和23.2%。结论：胞内Na⁺超载确可引起Na⁺-Ca²⁺交换系统激活;阿米洛利对豚鼠心室肌细胞I_Na-Ca有抑制作用,且对I_Na-Ca外向成分的抑制作用大于对内向成分的抑制作用。     

Increase in Na+−Ca2+ exchange activity in sarcolemma isolated from mesenteric arteries of spontaneously hypertensive rats

Na⁺?Ca²⁺ exchange process in sarcolemmal vesicles isolated from mesenteric arteries of Wistar-Kyoto normotensive(WKY) and spontaneously hypertensive rats(SHR) was investigated. The sarcolemmal fractions isolated after homogenization and sucrose density gradient centrifugation were enriched with 5′-nucleotidase and ouabain sensitive, K⁺-dependent phosphatase activities. When the vesicles were loaded with Na⁺, a time dependent Ca²⁺ uptake was observed. However, very little Ca²⁺ uptake was observed when the vesicles were loaded with K⁺, or Ca²⁺ uptake of the Na⁺-loaded vesicles was carried out in high sodium medium so that there was no sodium gradient. When the vesicles loaded with Ca²⁺ by Na⁺?Ca²⁺ exchange were diluted into potassium medium containing EGTA, Ca²⁺ was rapidly released from the vesicles. Na⁺-dependent Ca²⁺ uptake was increased in SHR compared to WKY, but passive efflux of preaccumulated Ca²⁺ from the vesicles was decreased in SHR. The data indicate that the membrane vesicles of rat mesenteric arteries exhibit Na⁺?Ca²⁺ exchange activity. It is also suggested that changes of this process in vascular smooth muscle cell membrane of SHR may be involved in higher intracellular Ca²⁺ concentration and higher basal tone in SHR.     

Characteristics of palytoxin-induced cation currents and Ca2+ mobilization in smooth muscle cells of rabbit portal vein

We examined the nature of the palytoxin (PTX)-induced channel and its relevance to the Ca²⁺ mobilizing effect of the toxin on smooth muscle cells isolated from rabbit portal vein using whole-cell voltage-clamp and microfluorimetric techniques. PTX (1 nM) induced a sustained, irreversible inward current at a holding potential of –40 mV. The PTX-induced current reversed at 0.5 ± 0.6 mV, and the PTX-induced channel permitted the passage of Na⁺, K⁺, Cs⁺ and, to a lesser extent, Li⁺, but not choline⁺ or Ca²⁺. During the sustained phase of the current, superfusion of Ni²⁺ (5 mM), La³⁺ (0.5 mM) or 2,4-dichlorobenzamil (2,4-DCB, 25 μM) reduced the current amplitude and decreased the slope conductance without changing the reversal potential. In 5 of 7 experiments, ouabain transiently increased the PTX-induced inward current and shifted the reversal potential in a positive direction. Subsequently, ouabain inhibited the current in every cell. PTX (10 nM) induced a sustained rise in cytosolic Ca²⁺ ([Ca²⁺]_i), which was resistant to verapamil but suppressed by omission of extracellular Ca²⁺. When external Na⁺ was replaced by choline⁺, PTX did not increase [Ca²⁺]_i. Pretreatment with 2,4-DCB prevented the elevation of [Ca²⁺]_i due to PTX. These results suggest that PTX does not directly stimulate Ca²⁺ entry but induces entry through Na⁺-Ca²⁺ exchange as a consequence of increased cytosolic Na⁺. Ni²⁺, La³⁺, 2,4-DCB and ouabain were shown to act as blockers of the PTX-induced channel. Ouabain may also inhibit Na⁺ pump current activated by cytosolic Na⁺. Received: 15 May 1996 / Accepted: 28 August 1996     

Sodium-Calcium Exchangers in Rat Ameloblasts

Although the central role of ameloblasts in synthesis and resorption of enamel matrix proteins during amelogenesis is well documented, the Ca²⁺-transport/extrusion mechanism remains to be fully elucidated. To clarify Ca²⁺-transport in rat ameloblasts, we investigated expression and localization of Na⁺-Ca²⁺ exchanger (NCX) isoforms and the functional characteristics of their ion transporting/pharmacological properties. RT-PCR and immunohistochemical analyses revealed expression of NCX1 and NCX3 in ameloblasts, localized in the apical membrane. In patch-clamp recordings, Ca²⁺ efflux by Na⁺-Ca²⁺ exchange showed dependence on external Na⁺. Ca²⁺ influx by Na⁺-Ca²⁺ exchange, measured by fura-2 fluorescence, showed dependence on extracellular Ca²⁺ concentration, and it was blocked by NCX inhibitors KB-R7943, SEA0400, and SN-6. These results showed significant expression of NCX1 and NCX3 in ameloblasts, indicating their involvement in the directional Ca²⁺ extrusion pathway from cells to the enamel mineralizing front.     

Neuropeptide Y as a stimulator of Na+-dependent Ca2+ efflux from freshly isolated adult rat cardiomyocytes

Several physiological stimuli cause a rise in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in cardiomyocytes. This increased [Ca²⁺]_i must be restored to physiological resting level to ensure response to further stimuli. In the present study, we examined the effect of neuropeptide Y (NPY), which is secreted from certain adrenergic or non-adrenergic neurons, on Ca²⁺ efflux from freshly isolated, quiescent adult rat cardiomyocytes. The isolated cardiomyocytes were preloaded with ⁴⁵CaCl₂ for 1 h. Then, the fractional release of ⁴⁵Ca²⁺ from the cells was measured. NPY stimulated the efflux of ⁴⁵Ca²⁺ from isolated adult rat cardiomyocytes in a concentration-dependent manner (10^–8 M to 10^–6 M). NPY (10^–6 M)-induced Ca²⁺ efflux was 2.0 ± 0.16% of the total cellular content. The ⁴⁵Ca²⁺ efflux from the cells was also stimulated by Y₁ receptor agonist, [Leu³¹, Pro³⁴]NPY, but not by Y₂ receptor agonist, NPY_13–36. The effect of NPY was inhibited by a peptide NPY inhibitor, NPY_18–36 and a non-peptide NPY inhibitor, benextramine to a similar extent. From these results, it is conceivable that the effect of NPY on Ca²⁺ efflux from cardiomyocytes is mediated through Y₁ receptors. It was also observed that NPY caused a rise in [Ca²⁺]_i to almost 150 nM. NPY-stimulated ⁴⁵Ca²⁺ efflux was not affected by removal of extracellular Ca²⁺, but was dependent on the presence of extracellular Na⁺. Moreover, NPY caused a ²²Na⁺ influx into the cells of about 1.6-fold over the basal value which was inhibited by amiloride and 5-(N,N-dimethyl)-amiloride, known Na⁺/Ca²⁺ exchange inhibitors. In addition, isoproterenol also caused ⁴⁵Ca²⁺ efflux from the cells and which was enhanced by the addition of NPY. These results suggest that NPY stimulates extracellular Na⁺-dependent ⁴⁵Ca²⁺ efflux from freshly isolated adult rat cardiomyocytes, probably through its stimulatory effect on plasma membrane Y₁ receptors with which NPY may couple during Na⁺/Ca²⁺ exchange. Received: 21 May 1997 / Accepted: 26 August 1997     

ION CHANNEL MODULATORS AS POTENTIAL POSITIVE INOTROPIC COMPOUNDS FOR TREATMENT OF HEART FAILURE

1. Current positive inotropy therapy of heart failure is associated with major problems: digoxin and the phosphodiesterase inhibitors can cause life-threatening toxicity while β-adrenoceptor agonists become less effective inotropic compounds as heart failure progresses. A new approach to positive inotropy is ion channel modulation. 2. An increased influx of Na⁺ during the cardiac action potential, as measured with DPI 201–106 and BDF 9148 which increase the probability of the open state of the Na⁺ channel, will increase force of contraction. 3. Activation of L-type Ca²⁺ channels with Bay K 8644 will increase influx of Ca²⁺ and increase the force of contraction. However the Ca²⁺ channel activators developed to date have little potential for the treatment of heart failure as they are vasoconstrictors. 4. Blocking cardiac K⁺ channels is a possible mechanism of positive inotropy. Terikalant inhibits the inward rectifying K⁺ channel, tedisamil inhibits the transient outward K⁺ channel and dofetilide is one of the newly developed inhibitors of the slow delayed outward rectifying K⁺ channel. All these drugs prolong the cardiac action potential to increase Ca²⁺ entry and force of contraction. 5. Thus drugs which increase Na⁺ influx or block K⁺ channels represent exciting possibilities for positive inotropy and the potential of these compounds for the treatment of heart failure needs to be fully evaluated.     

INTERACTIONS OF Na+, H2O2 AND THE Na+-Ca2+ EXCHANGER STIMULATE Ca2+ RELEASE IN CK1.4 CELLS

1. The present study aimed to demonstrate that interactions of cations, hydrogen peroxide (H₂O₂) and the Na⁺-Ca²⁺exchanger stimulate Ca²⁺ release and oscillations of cytosolic Ca²⁺ [Ca²⁺]i in non-transfected Chinese Hamster Ovary (CHO) C1 cells and in transfected CHO (CK1.4) cells that contained an expression vector coding the Na⁺-Ca²⁺ exchanger sequence. 2. The [⁴⁵Ca²⁺] uptake assay, fura-2 fluorescence imaging and 2² and 2³ factorial orthogonal statistics provide comparative, direct, efficient, quantitative and transient methods to delineate the effects of such interactions on Ca²⁺ influx, Ca²⁺release and [Ca²⁺]i in C1 and CK1.4 cells. 3. In contrast to the control of either Na⁺-, Ca²⁺- or H₂O₂-free or CI cells, an elevated [⁴⁵Ca²⁺] uptake was induced by Ca²⁺, Na⁺ and H₂O₂ individually and in combination, intracellular Ca²⁺ release was activated by H₂O₂ and by combinations of either H₂O₂ and Na⁺, H₂O₂ and the Na⁺-Ca²⁺ exchanger, Na⁺ and the Na⁺-Ca²⁺ exchanger or by H₂O₂, Na⁺ and the Na⁺-Ca²⁺ exchanger and a rise in [Ca²⁺]i was triggered by H₂O₂, Na⁺ and a combination of Na⁺ and the Na⁺-Ca²⁺exchanger. 4. These results indicate that interactions between H₂O₂, Na⁺ and the Na⁺-Ca²⁺ exchanger stimulate intracellular Ca²⁺mobilization via Ca²⁺-induced Ca²⁺ release mechanisms, ATP-activated G-protein coupled P_2y-purinoceptor-sensitive pathways, Na⁺-Ca²⁺ exchanger-mediated Ca²⁺ influx and cation-π interaction (a strong non-covalent force between the cation and the π face of an aromatic structure in the transmembrane protein). 5. The present findings provide important clues for understanding Ca²⁺ signal transduction mechanisms from the plasma membrane to the endoplasmic reticulum.     

INVOLVEMENT OF PROTEIN KINASE C IN THE REGULATION OF Na+/Ca2+ EXCHANGER IN BOVINE ADRENAL CHROMAFFIN CELLS

• 1 The Na⁺/Ca²⁺ exchanger (NCX) exchanges Na+ and Ca²⁺ bidirectionally through the forward mode (Ca²⁺ extrusion) or the reverse mode (Ca²⁺ influx). The present study was undertaken to clarify the role of protein kinase C (PKC) in the regulation of NCX in bovine adrenal chromaffin cells. The Na⁺‐loaded cells were prepared by treatment with 100 µmol/L ouabain and 50 µmol/L veratridine. Incubation of Na⁺‐loaded cells with Na⁺‐free solution in the presence of the Ca²⁺ channel blockers nicardipine (3 µmol/L) and ω‐conotoxin MVIIC (0.3 µmol/L) caused Ca²⁺ uptake and catecholamine release.
• 2 The Na⁺‐dependent Ca²⁺ uptake and catecholamine release were inhibited by 2‐[4‐[(2,5‐difluorophenyl)methoxy]phenoxy]‐5‐ethoxyaniline (SEA0400; 1 µmol/L) and 2‐[2‐[4‐(4‐nitrobenzyloxy)phenyl]isothiourea (KB‐R7943; 10 µmol/L), both NCX inhibitors. These results indicate that the Na⁺‐dependent responses are mostly due to activation of the NCX working in the reverse mode.
• 3 In addition, we examined the effects of PKC inhibitors and an activator on the NCX‐mediated Ca²⁺ uptake and catecholamine release. Bisindolylmaleimide I (0.3–10 µmol/L) and chelerythrine (3–100 µmol/L), both PKC inhibitors, inhibited NCX‐mediated responses. In contrast, phorbol 12,13‐dibutyrate (0.1–10 µmol/L), a PKC activator, enhanced the responses. Bisindolylmaleimide I and chelerythrine, at effective concentrations for inhibition of Na⁺‐dependent catecholamine release, had a little or no effect on high K⁺‐induced catecholamine release in intact cells or on Ca²⁺‐induced catecholamine release in β‐escin‐permeabilized cells.
• 4 These results suggest that PKC is involved in the activation of NCX in bovine adrenal chromaffin cells.

     

羟苯氨酮强心作用的生化机理研究

目的：研究羟苯氨酮(oxyphenamone, Oxy)强心作用的生化机理。方法：采用Na⁺,K⁺-ATP酶活性和cAMP-PDE活性、肌浆网Ca²⁺-ATP酶活性和cAMP含量以及心肌肌原纤维Ca²⁺,Mg²⁺-ATP酶活性等测定法,研究Oxy对它们的影响,并与milrinone和MCI-154作比较。 结果：Oxy对Na⁺,K⁺-ATP酶和PDE无抑制作用,也不影响心肌cAMP含量,但能显著增强心肌肌原纤维对Ca²⁺的敏感性,高浓度时轻度抑制心肌肌浆网Ca²⁺-ATP酶活性。结论：Oxy的强心作用方式不同于强心苷、β受体激动剂和PDE抑制剂等强心药,可能为一种新的钙增敏性强心药物。     

Effects of altered availability of Na+ on guinea-pig airway smooth muscle contractility

The effects of altering the availability of sodium ions (Na⁺) on contractility of the guinea-pig isolated trachealis was examined using regimens which are reported to inhibit Na⁺/K⁺ ATPase activity (ouabain), Na⁺/H⁺ exchange (amiloride, ammonium ion (NH₄⁺)) or Na⁺/Ca²⁺ exchange (reduced extracellular Na⁺). Inhibition of Na⁺/K⁺ ATPase and reversal of Na⁺/Ca²⁺ exchange resulted in increased ⁴⁵Ca uptake and contraction of the trachealis by voltage-sensitive and voltage-insensitive mechanisms respectively. When Na⁺/H⁺ exchange was inhibited by amiloride the tissues relaxed to below their baseline tension. The relaxation was not due to reduced Ca²⁺ influx. Treatment with NH₄⁺ produced a contractile response. Reduced extracellular Na⁺ caused a transient contraction as a result of reversal of the normal Na⁺/Ca²⁺exchange process leading to accumulation of Ca²⁺ within the cell. Since the effects of amiloride and reduced extracellular sodium were different, it is unlikely that amiloride is acting primarily by inhibiting Na⁺/Ca²⁺ exchange. Amiloride reduced tissue sensitivity to methacholine and KCI without affecting Ca²⁺ influx. This may involve a secondary stimulation of Na⁺/Ca²⁺ exchange following changes in [Na'];. Ouabain also reduced tissue sensitivity to methacholine and KCl.These findings suggest that Na⁺ are important in determining smooth muscle contractility. If NH₄⁺ is altering pH then, at the concentrations used, the changes in (H⁺] were not sufficient to alter responses to the spasmogens.     

Multiple effects of 1-[β-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF 96365) on Ca2+ signaling in MDCK cells: depletion of thapsigargin-sensitive Ca2+ store followed by capacitative Ca2+ entry, activation of a direct Ca2+ entry, and inhibition of thapsigargin-induced capacitative Ca2+ entry

The effect of 1-[β-[3-(4-methoxyphenyl)pro- poxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF 96365) on Ca²⁺ signaling in Madin Darby canine kidney (MDCK) cells was examined. SKF 96365 at 25–100 μM evoked a robust [Ca²⁺]_i transient in a dose-dependent manner, measured by fura-2 fluorimetry. A concentration of 10 μM SKF 96365 did not have an effect. The transient consisted of a slow rise, a gradual decay, and a sustained plateau in physiological Ca²⁺ medium. Removal of extracellular Ca²⁺ reduced the Ca²⁺ signals evoked by 50–100 μM SKF 96365 by nearly half in the area under the curve, suggesting that SKF 96365 induced intracellular Ca²⁺ release and also extracellular Ca²⁺ influx. A concentration of 100 μM SKF 96365 caused significant Mn²⁺ quench of fura-2 fluorescence, which was partly inhibited by La³⁺ (1 mM) or Gd³⁺ (0.1 mM), indicating that the SKF 96365-induced Ca²⁺ influx had two components: one is sensitive to La³⁺ (1 mM) or Gd³⁺ (0.1 mM), the other is not. The internal Ca²⁺ source for the SKF 96365-induced [Ca²⁺]_i transient was the endoplasmic reticulum Ca²⁺ store because, pretreatment with thapsigargin and cyclopiazonic acid, two inhibitors of the endoplasmic reticulum Ca²⁺ pump nearly abolished the SKF 96365-induced [Ca²⁺]_i increase in Ca²⁺-free medium. In contrast, pretreatment with 100 μM SKF 96365 only partly depleted the thapsigargin-sensitive Ca²⁺ store. Addition of 10 mM Ca²⁺ induced a significant [Ca²⁺]_i increase after prior incubation with 100 μM SKF 96365 in Ca²⁺-free medium, demonstrating that SKF 96365 induced capacitative Ca²⁺ entry. This capacitative Ca²⁺ entry was about 40% of that induced by 1 μM thapsigargin. Additional to inducing its own capacitative Ca²⁺ entry, 100 μM SKF 96365 partly inhibited thapsigargin- or uridine trisphos-phate (UTP)-induced capacitative Ca²⁺ entry. We also investigated the mechanisms underlying the decay of the SKF 96365-induced [Ca²⁺]_i transient. Inhibition of the plasma membrane Ca²⁺ pump with La³⁺ or Gd³⁺, or lowering extracellular Na⁺ level to 0.35 mM, significantly increased the SKF 96365-induced [Ca²⁺]_i transient. In contrast, the mitochondrial uncoupler carbonylcyanide m-chlorophenylhydrazone had little effect. In Ca²⁺-free medium, the thapsigargin-induced [Ca²⁺]_i increase was greatly reduced by pretreatment with SKF 96365. Collectively, we have found that besides its well-known inhibitory action on capacitative Ca²⁺ entry in many cell types, in MDCK cells SKF 96365 exerted multiple and complex effects on Ca²⁺ signaling. It induced a considerable increase in [Ca²⁺]_i by releasing Ca²⁺ from the endoplasmic reticulum store followed by capacitative Ca²⁺ entry. It also caused a direct Ca²⁺ entry. The decay of the SKF 96365 response was significantly governed by efflux via the plasma membrane Ca²⁺ pump or Na⁺/Ca²⁺ exchange. Sequestration by mitochondria or the endoplasmic reticulum played a minor role. We caution use of SKF 96365 as an inhibitor of capacitative Ca²⁺ entry. Received: 21 September 1998 / Accepted: 2 December 1998     

四肽FMRFa对大鼠心室肌Na+/Ca2+交换的抑制

目的　研究四肽FMRFa对大鼠单个心室肌细胞Na⁺/Ca²⁺交换的作用。方法　用膜片钳全细胞记录法测定成年大鼠心室肌细胞Na⁺/Ca²⁺交换电流(I_Na⁺/Ca²⁺)和其他离子通道电流。结果　FMRFa对大鼠心室肌细胞I_Na⁺/Ca²⁺呈浓度依赖性抑制,100μmol·L^-1浓度时抑制内向和外向I_Na⁺/Ca²⁺密度分别达60.1%和56.5%,对内向电流及外向电流的IC₅₀分别为20μmol·L^-1和34μmol·L^-1。FMRFa5μmol·L^-1抑制I_Na⁺/Ca²⁺内向和外向电流密度分别为38.7%和34.9%,但FMRFa5μmol·L^-1及20μmol·L^-1对L型钙电流、钠电流、瞬时外向电流和内向整流钾电流均无显著抑制作用。结论　FMRFa对大鼠心室肌细胞是一个特异性Na⁺/Ca²⁺交换抑制剂。     

Evidence against a regulation of Na+/K+-ATPase by Gi proteins. Failure to detect an influence of G proteins on Na+/Ca2+-exchange in cardiac sarcolemmal membranes

In the myocardium the inhibitory guanine nucleotide-binding regulatory proteins (G_i proteins) mediate negative chronotropic and negative inotropic effects by activation of K⁺ channels and inhibition of adenylyl cyclase. The concept of a uniform inhibitory action of G_i proteins on myocardial cellular activity has been questioned by the recent observations of adenosine-induced activation of the Na⁺/Ca²⁺ exchange and a carbachol-induced inhibition of the Na⁺/K⁺-ATPase activity in cardiac sarcolemmal membranes. The aim of the present study, therefore, was to reinvestigate the putative regulation of Na⁺/Ca²⁺ exchange and Na⁺/K⁺-ATPase activity in purified canine sarcolemmal membranes. These membranes were enriched in adenosine A₁ (Maximum number of receptors, B _max 0.033 pmol/mg) and muscarinic M₂ (B _max 2.9 pmol/mg) receptors and contained G_i2 and G_i3, two G_i protein isoforms, and Go, another pertussis toxin-sensitive G protein, as detected with specific antibodies. The adenosine A₁-selective agonist, (–)-N ⁶-(2-phenylisopropyl)-adenosine, and the muscarinic agonist, carbachol, both inhibited isoprenaline-stimulated adenylyl cyclase activity by 25% and 35% respectively, and the stable GTP analogue 5-guanylylimidodiphosphate inhibited forskolin-stimulated adenylyl cyclase activity by 35% in these membranes. The characteristics of Na⁺/Ca²⁺ exchange and Na⁺/K⁺-ATPase activity as well as those of the ouabain-sensitive, K⁺-activated 4-nitrophenylphosphatase, an ATP-independent, partial reaction of the Na⁺/K⁺-ATPase, were in agreement with published data with regard to specific activity, time course of activity and substrate dependency. However, none of these activities were influenced by adenosine, (–)-N ⁶-(2-phenylisopropyl)-adenosine, carbachol, or stable GTP analogs, suggesting that Na⁺/Ca²⁺ exchange and Na⁺/K⁺-ATPase are not regulated by Gi proteins in canine cardiac sarcolemmal membranes.     

Mechanisms of reduced contractility in an animal model of hypertensive heart failure

1. Alterations in intracellular Ca²⁺ homeostasis have frequently been implicated as underlying the contractile dysfunction of failing hearts. Contraction in cardiac muscle is due to a balance between sarcolemmal (SL) and sarcoplasmic reticulum (SR) Ca²⁺ transport, which has been studied in single cells and small tissue samples. However, many studies have not used physiological temperatures and pacing rates, and this could be problematic given different temperature dependencies and kinetics for transport processes. 2. Spontaneously‐hypertensive rats (SHR) and their age‐matched Wistar Kyoto controls (WKY) provide an animal model of hypertensive failure with many features in common to heart failure in humans. Steady‐state measurements of Ca²⁺ and force showed that peak stress was reduced in trabeculae from failing SHR hearts in comparison to WKY, although the Ca²⁺ transients were bigger and decayed more slowly. 3. Dynamic Ca²⁺ cycling was investigated by determining the recirculation fraction (RF) of activator Ca²⁺ through the SR between beats during recovery from experimental protocols that potentiated twitch force. No difference in RF between rat strains was found, although the RF was dependent on the potentiation protocol used. 4. Superfusion with 10 mmol/L caffeine and 0 mmol/L [Ca²⁺]_o was used to measure SL Ca²⁺ extrusion. The caffeine‐induced [Ca²⁺]_i transient decayed more slowly in SHR trabeculae, suggesting that SL Ca²⁺ extrusion was slower in SHR. 5. An ultrastructural immunohistochemical analysis of left ventricular free wall sections using confocal microscopy showed that t‐tubule organization was disrupted in myocytes from SHR, with reduced labelling of the SR Ca²⁺‐ATPase and Na⁺–Ca²⁺ exchanger in comparison to WKY, with the latter possibly related to a lower fraction of t‐tubules per unit cell volume. 6. We suggest that although Ca²⁺ transport is altered in the progression to heart failure, force development is not limited by the amplitude of the Ca²⁺ transient. Despite slower SR Ca²⁺ transport, the recirculation fraction and dynamic response to a change of inotropic state minimally altered changes in the SHR model because there was a similar slowing in Ca²⁺ extrusion across the surface membrane.     

Functional expression of voltage-gated Na+ and Ca2+ channels during neuronal differentiation of PC12 cells with nerve growth factor or forskolin

Voltage-gated ion channels and morphological differentiation were studied in rat PC12 pheochromocytoma cells after treatment with nerve growth factor (NGF) or forskolin. Ca²⁺ and Na⁺ channels were analyzed by electrophysiological techniques (using Ba²⁺ as charge carrier through Ca²⁺ channels) and by binding studies with specific ligands. With NGF, Na⁺ current (I _Na) density increased in parallel with neurite extension. Ba²⁺ current (I _Ba) density and Ca²⁺ channel numbers were both enhanced after a 2-day latency period. The tyrosine kinase inhibitor genistein blocked NGF-induced neurite extension but not the increase in I _Na density. With forskolin, neurite outgrowth was linked to an apparent increase in I _Ba density similar to the one induced by NGF, while no change in I _Na was observed. Dihydropyridine-sensitive (L-type) as well as ω-conotoxin-sensitive (N-type) currents contributed to this effect. In spite of its stimulating effect on I _Ba, binding studies with radiolabeled ligands in forskolin-treated cells showed no change in N-type and an apparent loss of high affinity L-type Ca²⁺ channel binding. Our results suggest that induction of individual voltage-dependent channel types as well as morphological differentiation each require the activation of different signaling pathways. NGF and forskolin both enhanced current flow through voltage-dependent Ca²⁺ channels. However, only NGF increased channel expression while forskolin appeared to modulate channel kinetics. Received: 15 December 1998 / Accepted: 15 February 1999     

Pharmacological differentiation of synergistic contribution of L-type Ca2+ channels and Na+/H+ exchange to the positive inotropic effect of phenylephrine,endothelin-3 and angiotensin II in rabbit ventricular myocardium

The present study was designed to delineate pharmacologically the role of sarcolemmal L-type Ca²⁺ channels and Na⁺/H⁺ exchange in the positive inotropic effect (PIE) of phenylephrine mediated by alpha-1 adrenoceptors, endothelin (ET) and angiotensin II (Ang II) that stimulate phosphoinositide (PI) hydrolysis in the rabbit ventricular muscle. The PIE of these receptor agonists was compared with the PIE of isoprenaline that accumulates cyclic AMP. For this purpose, we investigated the influence of a Ca²⁺ antagonist, verapamil, and of an inhibitor of Na⁺/H⁺ exchange, 5-(N-ethyl-N-isopropyl) amiloride (EIPA), alone or in combination, on the cumulative concentration-response curve (CRC) for phenylephrine (with 0.3 μM bupranolol), ET-3 and Ang II in isolated right ventricular papillary muscles of the rabbit, which were electrically stimulated at 1 Hz in Krebs-Henseleit solution at 37°C. Verapamil at 0.3 and 1 μM decreased the basal force of contraction to 37.0 ± 4.0% and 13.2 ± 1.1% of the control, respectively, while EIPA even at 10 μM affected the basal force to much less extent and decreased it to 87.0 ± 1.4%. Verapamil (0.3 and 1 μM) and EIPA (1 and 10 μM), when used alone, each significantly attenuated but did not abolish the PIEs induced by phenylephrine, ET-3 and Ang II, while the simultaneous administration of verapamil (1 μM) and EIPA (10 μM) consistently and almost completely inhibited the PIE induced by these receptor agonists. By contrast, the PIE of isoprenaline was retained even in the presence of verapamil and EIPA. These results indicate that both the influx of Ca²⁺ ions through L-type Ca²⁺ channels and activation of Na⁺/H⁺ exchange contribute synergistically to the PIE that is mediated by alpha-1 adrenergic, ET and Ang II receptor agonists, while these mechanisms are not essential for the beta-adrenoceptor-mediated PIE. Received: 20 February 1996 / Accepted: 20 August 1996     

Inhibition of Sarcolemma ATPases by some Membrane-stabilizing Drugs

Abstract: Rat sarcolemma preparations were incubated with some membranes stabilizers to study their effects on the (Na⁺,K⁺)- and Ca²⁺-ATPase activity. The drugs inhibited the enzymes with the same order of potency as in the earlier observed muscle contractures: chlorpromazine> dibucaine> propranolol> tetracaine> procaine (range 0.1–3.6 mM) (Røed & Brodal 1979). The contracture inducing effect of the stabilizers is suggested to be caused by a membrane depolarization due to the (Na⁺,K⁺)-ATPase inhibition. Ouabain inhibited the (Na⁺,K⁺)-ATPase activity in purified plasma membrane, but did not inhibit the sarcolemma located ATPases or induce any contracture.     

N,N-dimethyl-D-erythro-sphingosine increases intracellular Ca2+ concentration via Na+-Ca2+-exchanger in HCT116 human colon cancer cells

N,N-dimethyl-D-erythro-sphingosine (DMS), an N-methyl derivative of sphingosine, is an inhibitor of protein kinase C (PKC) and sphingosine kinase (SK). In previous reports, DMS-induced intracellular Ca²⁺ increase concentration ([Ca²⁺]_i) was studied in T lymphocytes, monocytes, astrocytes and neuronal cells. In the present study, we studied DMS-induced increase of [Ca²⁺]_i in HCT116 human colon cancer cells. We found that the DMS-induced increase of [Ca²⁺]_i in colon cancer cells is composed of Ca²⁺ release from intracellular Ca²⁺ stores and subsequent Ca²⁺ influx. The Ca²⁺ release is not related to modulation of inositol 1,4,5-trisphosphate (IP₃) receptor or ryanodine receptor. On the other hand, the Ca²⁺ influx is mediated largely through Ca²⁺ channels sensitive to verapamil, nifedipine, Ga³⁺, and La³⁺. Furthermore, we found that the response is inhibited by bepridil and Ni²⁺, specific inhibitors of Na⁺-Ca²⁺-exchanger, suggesting involvement of Na⁺-Ca²⁺ exchanger in the DMS-induced [Ca²⁺]_i increase in colon cancer cells. This inhibition was also observed in U937 monocytes, but not in 1321N1 astrocytes.     

ROLE OF EXTRACELLULAR Na+, Ca2+-ACTIVATED Cl- CHANNELS AND BK CHANNELS IN THE CONTRACTION OF Ca2+ STORE-DEPLETED TRACHEAL SMOOTH MUSCLE

• 1 In the present study, we investigated the series of events involved in the contraction of tracheal smooth muscle induced by the re‐addition of Ca²⁺ in an in vitro experimental model in which Ca²⁺ stores had been depleted and their refilling had been blocked by thapsigargin.
• 2 Mean (±SEM) contraction was diminished by: (i) inhibitors of store‐operated calcium channels (SOCC), namely 100  µ mol/L SKF‐96365 and 100  µ mol/L 1‐(2‐trifluoromethylphenyl) imidazole (to 66.3 ± 4.4 and 41.3 ± 5.2% of control, respectively); (ii) inhibitors of voltage‐gated Ca²⁺ channels Ca_V1.2 channels, namely 1  µ mol/L nifedipine and 10  µ mol/L verapamil (to 86.2 ± 3.4 and 76.9 ± 5.9% of control, respectively); and (iii) 20  µ mol/L niflumic acid, a non‐selective inhibitor of Ca²⁺‐dependent Cl^? channels (to 41.1 ± 9.8% of control). In contrast, contraction was increased 2.3‐fold by 100 nmol/L iberiotoxin, a blocker of the large‐conductance Ca²⁺‐activated K⁺ (BK) channels.
• 3 Furthermore, contraction was significantly inhibited when Na⁺ in the bathing solution was replaced by N‐methyl–d ‐glucamine (NMDG⁺) to 39.9 ± 7.2% of control, but not when it was replaced by Li⁺ (114.5 ± 24.4% of control). In addition, when Na⁺ had been replaced by NMDG⁺, contractions were further inhibited by both nifedipine and niflumic acid (to 3.0 ± 1.8 and 24.4 ± 8.1% of control, respectively). Nifedipine also reduced contractions when Na⁺ had been replaced by Li⁺ (to 10.7 ± 3.4% to control), the niflumic acid had no effect (116.0 ± 4.5% of control).
• 4 In conclusion, the data of the present study demonstrate the roles of SOCC, BK channels and Ca_V1.2 channels in the contractions induced by the re‐addition of Ca²⁺ to the solution bathing guinea‐pig tracheal rings under conditions of Ca²⁺‐depleted sacroplasmic reticulum and inhibition of sarcoplasmic/endoplasmic reticulum calcium ATPase. The contractions were highly dependent on extracellular Na⁺, suggesting a role for SOCC in mediating the Na⁺ influx.

     

Long-term nifedipine treatment reduces calcium overload in isolated reperfused hearts of diabetic rats

• 1.1. Streptozotocin-induced diabetic rats showed poor post-ischemic recovery in isolated working rat hearts.
• 2.2. Diabetic rats showed myocardial Na⁺ accumulation after ischemia, and Ca²⁺ level and water content elevation after reperfusion.
• 3.3. A 6-wk nifedipine treatment improved post-ischemic recovery of cardiac parameters and prevented myocardial Na⁺ accumulation after ischemia and myocardial Ca²⁺ level and water content elevation after reperfusion of diabetic rats.
• 4.4. Results suggest that nifedipine treatment improves cardiac dysfunction in the reperfused ischemic hearts of diabetic rats through normalization of the Na⁺-Ca²⁺ imbalance and water content.