Multiple transport modes of the cardiac Na+/Ca2+ exchanger

The cardiac Na+/Ca2+ exchanger (NCX1; ref. 2) is a bi-directional Ca2+ transporter that contributes to the electrical activity of the heart. When, and if, Ca2+ is exported or imported depends on the Na+/Ca2+ exchange ratio. Whereas a ratio of 3:1 (Na+:Ca2+) has been indicated by Ca2+ flux equilibrium studies, a ratio closer to 4:1 has been indicated by exchange current reversal potentials. Here we show, using an ion-selective electrode technique to quantify ion fluxes in giant patches, that ion flux ratios are approximately 3.2 for maximal transport in either direction. With Na+ and Ca2+ on both sides of the membrane, net current and Ca2+ flux can reverse at different membrane potentials, and inward current can be generated in the absence of cytoplasmic Ca2+, but not Na+. We propose that NCX1 can transport not only 1 Ca2+ or 3 Na+ ions, but also 1 Ca2+ with 1 Na+ ion at a low rate. Therefore, in addition to the major 3:1 transport mode, import of 1 Na+ with 1 Ca2+ defines a Na+-conducting mode that exports 1 Ca2+, and an electroneutral Ca2+ influx mode that exports 3 Na+. The two minor transport modes can potentially determine resting free Ca2+ and background inward current in heart.     

Na-Ca exchange current in mammalian heart cells

Electrogenic Na-Ca exchange has been known to act in the cardiac sarcolemma as a major mechanism for extruding Ca ions. Ionic flux measurements in cardiac vesicles have recently suggested that the exchange ratio is probably 3 Na:1 Ca, although a membrane current generated by such a process has not been isolated. Using the intracellular perfusion technique combined with the whole-cell voltage clamp, we were able to load Na+ inside and Ca2+ outside the single ventricular cells of the guinea pig and have succeeded in recording an outward Na-Ca exchange current while blocking most other membrane currents. The current is voltage-dependent, blocked by La3+ and does not develop in the absence of intracellular free Ca2+. This report presents the first direct measurement of the cardiac Na-Ca exchange current, and should facilitate the study of Ca2+ fluxes during cardiac activity, together with various electrical changes attributable to the Na-Ca exchange and the testing of proposed models.     

Identification of Na-Ca exchange current in single cardiac myocytes

In cardiac muscle the exchange of intracellular Ca2+ for extracellular Na+ is an important transport mechanism for regulation of the intracellular free Ca2+ concentration [( Ca]i) and hence the contractile strength of the heart. Due to its stoichiometry of greater than or equal to 3:1 Na+/Ca2+ (refs 3,5), Na-Ca exchange is supposed to generate a current across the cell membrane. It is thought that such a current may contribute to cardiac action potential and physiological or pathological pacemaker activity. Although the occurrence of Na-Ca exchange is well documented, a membrane current generated by this transport has not been identified unequivocally. Previous attempts to detect such a current in multicellular preparations, for example, by measuring small current differences after varying the extracellular ionic composition, although providing evidence, did not rule out other possible interpretations. Here we demonstrate that a transient rise in [Ca]i caused by release of Ca from sarcoplasmic reticulum (SR) generates a membrane current in cardiac myocytes. The dependence of this current on the transmembrane gradients for Na+ and Ca2+ and on membrane potential meets the criteria for a current produced by electrogenic Na-Ca exchange. Cyclic activation of this current by release of Ca from the SR can cause maintained spontaneous activity, suggesting that Na-Ca exchange contributes to certain forms of cardiac pacemaking.     

Extrusion of calcium from rod outer segments is driven by both sodium and potassium gradients

Calcium is transported across the surface membrane of both nerve and muscle by a Na+-dependent mechanism, usually termed the Na:Ca exchange. It is well established from experiments on rod outer segments that one net positive charge enters the cell for every Ca2+ ion extruded by the exchange, which is generally interpreted to imply an exchange stoichiometry of 3 Na+:1 Ca2+. We have measured the currents associated with the operation of the exchange in both forward and reversed modes in isolated rod outer segments and we find that the reversed mode, in which Ca2+ enters the cell in exchange for Na+, depends strongly on the presence of external K+. The ability of changes in external K+ concentration ([K+]o) to perturb the equilibrium level of [Ca2+]i indicates that K+ is co-transported with calcium. From an examination of the relative changes of [Ca2+]o, [Na+]o, [K+]o and membrane potential required to maintain the exchange at equilibrium, we conclude that the exchange stoichiometry is 4 Na+:1 Ca2+, 1 K+ and we propose that the exchange should be renamed the Na:Ca, K exchange. Harnessing the outward K+ gradient should allow the exchange to maintain a Ca2+ efflux down to levels of internal [Ca2+] that are considerably lower than would be possible with a 3 Na+:1 Ca2+ exchange.     

Molecular operations of the sodium-calcium exchanger revealed by conformation currents

The sodium-calcium exchanger is critical in the normal functioning of many cells. In heart muscle, it is the principal way by which the cells keep the concentration of intracellular calcium low, pumping out the Ca2+ that enters the cytosol through L-type Ca2+ channels. The exchanger may also contribute to the triggering of Ca2+ release during voltage-activated excitation-contraction coupling in heart. Time resolved examination of the conformational changes of macromolecules in living cells has so far been largely restricted to ion-channel proteins whose gating is voltage-dependent. We have now directly measured electrical currents arising from the molecular rearrangements of the sarcolemmal Na-Ca exchanger. Changes in the conformation of the exchanger protein were activated by a rapid increase in the intracellular calcium concentration produced by flash photolysis of caged calcium in voltage-clamped heart cells. Two components of membrane current were produced, reflecting a calcium-dependent conformational change of the transporter proteins and net transport of ions by the exchanger. The properties of these components provide evidence that the Na-Ca exchanger protein undergoes two consecutive membrane-crossing molecular transitions that each move charge, and that there are at least 250 exchangers per micron 2 turning over up to 2,500 times per second.     

Calcium-dependent regulation of cyclic GMP phosphodiesterase by a protein from frog retinal rods.

In vertebrate photoreceptors, light reduces cyclic GMP concentration and closes cGMP-activated channels to induce a hyperpolarizing response. As Ca2+ can permeate the channels and the Na(+)-Ca2+ exchanger continuously extrudes Ca2+, closure of the channel results in a reduction of the inter-rod Ca2+ concentration. This is believed to be one of the mechanisms of light-adaptation produced by activation of guanylate cyclase. Effects of Ca2+ on the cGMP phosphodiesterase (PDE) have been reported, but their physiological significance has remained unclear. We have perfused the inside-out preparation of a frog rod outer segment (I/O ROS, originally termed truncated ROS, and find that Ca2+ in a physiological range regulates the light-activation of PDE. Therefore, PDE regulation by Ca2+ must be involved in light-adaptation in rods. The effect is mediated by a newly found protein which binds to disk membranes at high Ca2+ concentrations and prolongs PDE activation.     

Charge movement during Na+ translocation by native and cloned cardiac Na+/Ca2+ exchanger

Na+/Ca2+ exchange is electrogenic and moves one net positive charge per cycle. Although the cardiac exchanger has a three-to-one Na+/Ca2+ stoichiometry, details of the reaction cycle are not well defined. Here we associate Na+ translocation by the cardiac exchanger with positive charge movement in giant membrane patches from cardiac myocytes and oocytes expressing the cloned cardiac Na+/Ca2+ exchanger. The charge movements are initiated by step increments of the cytoplasmic Na+ concentration in the absence of Ca2+. Giant patches from control oocytes lack both steady-state Na+/Ca2+ exchange current (INaCa) and Na(+)-induced charge movements. Charge movements indicate about 400 exchangers per micron 2 in guinea-pig sarcolemma. Fully activated INaCa densities (20-30 microA cm-2) indicate maximum turnover rates of 5,000 s-1. As has been predicted for consecutive exchange models, the apparent ion affinities of steady state INaCa increase as the counterion concentrations are decreased. Consistent with an electroneutral Ca2+ translocation, we find that voltage dependence of INaCa in both directions is lost as Ca2+ concentration is decreased. The principal electrogenic step seems to be at the extracellular end of the Na+ translocation pathway.     

Light-induced reduction of cytoplasmic free calcium in retinal rod outer segment

The response of retinal rod photoreceptors to light consists of a membrane hyperpolarization resulting from the decrease of a light-sensitive conductance in the outer segment. According to the calcium hypothesis, this conductance is blocked by a rise in intracellular free Ca triggered by light, a notion supported by the findings that an induced rise in internal Ca leads to blockage of the light-sensitive conductance and that light triggers a net Ca efflux from the outer segment via a Na-Ca exchanger, suggesting a rise in internal free Ca in the light. We have now measured both Ca influx and efflux through the outer segment plasma membrane and find that, contrary to the calcium hypothesis, light seems to decrease rather than increase the free Ca concentration in the rod outer segment. This result implies that Ca does not mediate visual excitation but it probably has a role in light adaptation.     

金属离子对紫叶小檗色素稳定性的影响

本文报道了几种常见金属离子对紫叶小檗色素稳定性的影响。结果表明：钠、钙、锌、铜、镁、锰、铝离子对紫叶小檗色素有增色,护色作用,铅离子对呈色无明显影响,但可加速色素的降解。铁、锡离子可使色素溶液变色。     

Effects of ATP and vanadate on calcium efflux from barnacle muscle fibres

Calcium ions carry the inward current during depolarization of barnacle muscle fibres and are involved in the contraction process. Intracellular ionized calcium ([Ca2+]i) in barnacle muscle, as in other cells, is kept at a very low concentration, against a large electrochemical gradient. This large gradient is maintained by Ca2+ extrusion mechanisms. When [Ca2+]i is below the contraction threshold, Ca2+ efflux from giant barnacle muscle fibres is, largely, both ATP dependent and external Na+ (Na+0) dependent (see also refs 5,6). When [Ca2+]i is raised to the level expected during muscle contraction (2-5 muM), most of the Ca2+ efflux from perfused fibres is Na0 dependent; as in squid axons, this Na+0-dependent Ca2+ efflux is ATP independent. Orthovanadate is an inhibitor of (Na+ + K+) ATPase and the red cell Ca2+-ATpase. We report here that vanadate inhibits ATP-promoted, Na+0-dependent Ca2+ efflux from barnacle muscle fibres perfused with low [Ca2+]i (0.2-0.5 microM), but has little effect on the Na+0-dependent, ATP-independent Ca2+ efflux from fibres with a high [Ca]i (2-5 microM). Nevertheless, ATP depletion or vanadate treatment of high [Ca2+]i fibres causes an approximately 50-fold increase of Ca2+ efflux into Ca2+-containing lithium seawater. These results demonstrate that both vanadate and ATP affect Ca2+ extrusion, including the Na+0-dependent Ca2+ efflux (Na-Ca exchange), in barnacle muscle.     

A novel receptor-operated Ca2+-permeable channel activated by ATP in smooth muscle

Receptor-operated Ca2+ entry has been proposed as a signalling mechanism in many cells. Receptor-operated Ca2+ channels (ROCs) were first postulated in smooth muscle by Bolton, van Breemen and Somlyo and Somlyo, but recordings of directly ligand-gated Ca2+ current are lacking. Here we describe receptor-operated Ca2+ current evoked in arterial smooth muscle cells by ATP, a sympathetic neurotransmitter. ATP activates channels with approximately 3:1 selectivity for Ca2+ over Na+ at near-physiological concentrations and with a unitary conductance of approximately 5 pS in 110 mM Ca2+ or Ba2+. The channels can be opened even at very negative potentials and resist inhibition by cadmium or nifedipine, unlike voltage-gated Ca2+ channels; they are not blocked by Mg2+, unlike NMDA (N-methyl-D-aspartate)-activated channels; they are directly activated by ligand, without involvement of readily diffusible second messengers, unlike cation channels in neutrophils and T lymphocytes. Thus, the ATP-activated channels provide a distinct mechanism for excitatory synaptic current and Ca2+ entry in smooth muscle.     

Control of Ca2+ in rod outer segment disks by light and cyclic GMP

Photons absorbed in vertebrate rods and cones probably cause electrochemical changes at the photoreceptor plasma membrane by changing the cytoplasmic concentration of a diffusible transmitter substance, reducing the Na+ current flowing into the outer segment of the cell in the dark, to produce the observed membrane hyperpolarization that is the initial excitatory response. Cyclic GMP has been proposed as the transmitter because a light-activated cyclic GMP phosphodiesterase (PDE) has been found in rod disk membranes and because intracellularly injected cyclic GMP reduces rod membrane potentials. Free Ca2+ has also been proposed because increasing external [Ca2+] quickly and reversibly reduces the dark current and divalent cationophores increase the Ca2+ sensitivity. Ca2+ efflux from rod outer segments (ROS) of intact retinas occurs simultaneously with light responses. Vesicles prepared from ROS disk membranes become more permeable on illumination, releasing trapped ions or molecules, but intact outer segment disks have not previously been found to store sufficient Ca2+ in darkness and to release enough in light to meet the theoretical requirements for control of the dark current by varying cytoplasmic Ca2+ (refs 14-18). We now report experiments that show the required Ca2+ storage and release from rod disk membranes suspended in media containing high-energy phosphate esters and electrolytes approximating the cytoplasmic composition of live rod cells. Cyclic GMP stimulates Ca2+ uptake by ROS disks in such media.     

新型螯合树脂的合成及其吸附性能研究

采用新型大孔政治协商会议状聚氯乙烯为骨架的多乙烯多胺树脂在盐酸存在下与亚磷酸、甲醛反应，合成了氨基酸型螯合树脂，研究了该螯合树脂对Ｃｕ＾２＋、Ｚｎ＾２＋、Ｃａ＾２＋、Ｍｇ＾２＋、Ｎａ＾２＋等离子的吸附率分别达９６．２％和９３．４％，对Ｃａ＾２＋、Ｍｇ＾２＋、Ｎａ＾＋等的吸附容量较低，对Ｃｕ＾２＋Ｚｎ＾２＋具有较好的吸附选择性，能分别从Ｃｕ＾２＋、Ｍｇ＾２＋、Ｃａ＾２＋、Ｎａ＾＋的混合离子中定量地吸     

Kinetics, stoichiometry and role of the Na-Ca exchange mechanism in isolated cardiac myocytes

Compelling evidence has existed for more than a decade for a sodium/calcium (Na-Ca) exchange mechanism in the surface membrane of mammalian heart muscle cells which exchanges about three sodium ions for each calcium ion. Although it is known that cardiac muscle contraction is regulated by a transient increase in intracellular calcium ([Ca2+]i) triggered by the action potential, the contribution of the Na-Ca exchanger to the [Ca2+]i transient and to calcium extrusion during rest is unclear. To clarify these questions, changes in [Ca2+]i were measured with indo-1 in single cardiac myocytes which were voltage clamped and dialysed with a physiological level of sodium. We find that Ca entry through the Na-Ca exchanger is too slow to affect markedly the rate of rise of the normal [Ca2+]i transient. On repolarization, Ca extrusion by the exchanger causes [Ca2+]i to decline with a time constant of 0.5 s at -80 mV. The rate of decline can be slowed e-fold with a 77-mV depolarization. Calcium extrusion by the exchanger can account for about 15% of the rate of decline of the [Ca2+]i transient (the remainder being calcium resequestration by the sarcoplasmic reticulum (SR]. The ability of the cell to extrude calcium was greatly reduced on inhibiting the exchanger by removing external sodium, which itself led to an increase in resting [Ca2+]i. This finding is in contrast to the suggestion that calcium extrusion at rest is mediated mainly by a sarcolemmal Ca-ATPase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Study of transmembrane La3+ movement in rat ventricular myocytes by the patch-clamp technique

We have studied transmembrane La3+ movement in rat ventricular myocytes for the first time by using the whole-cell patch-clamp recording mode. La3+ (0.01-5.0 mmol/L) could not bring out inward currents through the L-type calcium channel in rat ventricular myocytes, while it could enter the cells by the same way carried by 1μmol/L ionomycin. When the outward Na+ concentration gradient is formed, La3+ can enter the cells via Na-Ca exchange, and the exchange currentsincrease with the increase of external La3+ concentrations. But compared with Na-Ca exchange currents in the same concentration, the former is only 14%-38% of the latter. The patch-clamp experiment indicates that La3+ normally can not enter ventricular myocytes through L-type calcium channel, but it can enter the cells via Na-Ca exchange.     

Depletion of intracellular calcium stores activates a calcium current in mast cells.

In many cell types, receptor-mediated Ca2+ release from internal stores is followed by Ca2+ influx across the plasma membrane. The sustained entry of Ca2+ is thought to result partly from the depletion of intracellular Ca2+ pools. Most investigations have characterized Ca2+ influx indirectly by measuring Ca(2+)-activated currents or using Fura-2 quenching by Mn2+, which in some cells enters the cells by the same influx pathway. But only a few studies have investigated this Ca2+ entry pathway more directly. We have combined patch-clamp and Fura-2 measurements to monitor membrane currents in mast cells under conditions where intracellular Ca2+ stores were emptied by either inositol 1,4,5-trisphosphate, ionomycin, or excess of the Ca2+ chelator EGTA. The depletion of Ca2+ pools by these independent mechanisms commonly induced activation of a sustained calcium inward current that was highly selective for Ca2+ ions over Ba2+, Sr2+ and Mn2+. This Ca2+ current, which we term ICRAC (calcium release-activated calcium), is not voltage-activated and shows a characteristic inward rectification. It may be the mechanism by which electrically nonexcitable cells maintain raised intracellular Ca2+ concentrations and replenish their empty Ca2+ stores after receptor stimulation.     

Phorbol ester and diacylglycerol mimic growth factors in raising cytoplasmic pH

There is now good evidence that cytoplasmic pH (pHi) may have an important role in the metabolic activation of quiescent cells. In particular, growth stimulation of mammalian fibroblasts leads to a rapid increase in pHi (refs 3-6), due to activation of a Na+/H+ exchanger in the plasma membrane, and this alkalinization is necessary for the initiation of DNA synthesis. However, the mechanism by which mitogens activate the Na+/H+ exchanger to raise pHi is not known, although an increase in cytoplasmic free Ca2+ ([Ca2+]i) has been postulated as the primary trigger. We now present data suggesting that the Na+/H+ exchanger is set in motion through protein kinase C, a phospholipid- and Ca2+-dependent enzyme normally activated by diacylglycerol produced from inositol phospholipids in response to external stimuli. Using newly developed pH microelectrodes and fluorimetric techniques, we show that a tumour promoting phorbol ester and synthetic diacylglycerol, both potent activators of kinase C (refs 12-15), mimic the action of mitogens in rapidly elevating pHi in different cell types. Furthermore, we demonstrate that, contrary to previous views, an early rise in [Ca2+]i is not essential for the activation of Na+/H+ exchange and the resultant increase in pHi. Finally, we suggest that an alkaline pHi shift, mediated by Na+/H+ exchange, may be a common signal in the action of those hormones which elicit the breakdown of inositol phospholipids.     

Gelsolin inhibition of fast axonal transport indicates a requirement for actin microfilaments

The actions of actin-based microfilaments in cell motility suggest a possible role in the mechanism of fast axonal transport, but the pharmacological data evaluating their role in this process are equivocal. Moreover, microfilaments are difficult to preserve and identify in ultrastructural studies, so the organization and function of axonal actin has remained uncertain. We have now evaluated the role of actin microfilaments in intracellular transport of membranous organelles using video-enhanced contrast microscopy and gelsolin to analyse fast axonal transport directly in isolated axoplasm from the squid giant axon. With this preparation it is possible to perfuse axoplasm with large molecules that do not cross the plasmalemma, while controlling cation levels. The 90,000-molecular weight protein gelsolin depolymerizes actin microfilaments in micromolar Ca2+, but not in the absence of Ca2+. Axonal transport of membranous organelles has previously been shown to be unaffected by levels of Ca2+ up to 10 microM. In the presence of EGTA, gelsolin has no effect on the movement of membranous organelles, but in the presence of 10 microM Ca2+ it completely blocks transport of all membranous organelles. No changes in the organization of the axoplasm were detected. These results and results using other probes for actin are consistent with the hypothesis that actin-based microfilaments are involved in the movement of membranous organelles in the axon.     

Naloxone-reversible effect of opioids on pinocytosis in Amoeba proteus

A characteristic feature of induced pinocytosis in Amoeba proteus is the formation of broad channels by invagination of the cell membrane. This process, which requires Ca2+, occurs in response to depolarising cations. High Ca2+ levels reduce pinocytosis induced by cations such as Na+ and Tris+, whereas pinocytosis induced by K+ is less affected by Ca2+ (ref. 4). Agents which interfere with the calcium metabolism of the amoeba will therefore either stimulate or inhibit pinocytosis induced by Na+ (ref. 5). Among the agents which are supposed to reduce Ca2+ influx across cell membranes or otherwise decrease cellular availability of Ca2+ are the opiates and opioid peptides, high doses of which have been reported to affect the amoeba. Accordingly, Met-enkephalin, morphine and codeine potentiate the inhibition of pinocytosis caused by Ca2+-binding agents and reverse the calcium blockade of pinocytosis mediated by caffeine. In this report we show that pinocytosis induced by Na+ or Tris+ is suppressed by beta-endorphin, Metenkephalin and morphine. These effects were abolished or diminished by an opiate receptor antagonist, (-)naloxone, by increasing the Na+ concentration, or by addition of Ca2+.     

采出污水配制聚合物溶液的影响因素研究

研究了聚合物驱采出污水中Na+、K+、Ca2+、Mg2+、Fe2+、Fe3+六种离子对聚合物溶液初始黏度的影响和溶解氧、阳离子、粘土矿物以及细菌对其稳定性的影响程度,提出了采出污水配注聚合物的参考控制指标和限度。研究结果表明,高价金属阳离子是影响聚合物水溶液初始黏度的主要因素,其由大到小的顺序为Fe2+＞Fe3+＞Mg2+＞Ca2+＞Na+＞K+。配制用水的基本要求是不含铁离子,Ca2+离子含量控制在200mg/L以下,最好控制在50mg/L以下;Mg2+离子含量控制在100mg/L以下,最好控制在30mg/L以下;Na+、K+离子含量应该控制在2000mg/L以下,最好控制在500mg/L以下。高价金属离子和溶解氧是影响聚合物溶液黏度稳定性的主要因素,其由大到小的顺序为：Fe2+>Mg2+(Ca2+)>Fe3+>Na+(K+),一价阳离子和悬浮物对聚合物溶液黏度稳定性也有一定影响,细菌对其影响相对较小。