Oscillations of intracellular Ca2+ in mammalian cardiac muscle

Contraction of cardiac muscle depends on a transient rise of intracellular calcium concentration ([Ca2+]i) which is initiated by the action potential. It has, however, also been suggested that [Ca2+]i can fluctuate in the absence of changes in membrane potential. The evidence for this is indirect and comes from observations of (1) fluctuations of contractile force in intact cells, (2) spontaneous cellular movements, and (3) spontaneous contractions in cells which have been skinned to remove the surface membrane. The fluctuations in force are particularly prominent when the cell is Ca2+-loaded, and have been attributed to a Ca2+-induced Ca2+ release from the sarcoplasmic reticulum. In these conditions of Ca2+-loading the normal cardiac contraction is followed by an aftercontraction which has been attributed to the synchronization of the fluctuations. The rise of [Ca2+]i which is thought to underlie the aftercontraction also produces a transient inward current. This current, which probably results from a Ca2+-activated nonspecific cation conductance, has been implicated in the genesis of various cardiac arrhythmias. However, despite the potential importance of such fluctuations of [Ca2+]i their existence has, so far, only been inferred from tension measurements. Here we present direct measurements of such oscillations of [Ca2+]i.     

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.     

Glucagon stimulates the cardiac Ca2+ current by activation of adenylyl cyclase and inhibition of phosphodiesterase

Glucagon exerts positive inotropic and chronotropic effects in the heart. Like its glycogenolytic effect in liver cells, the cardiac effects of glucagon are often correlated with adenylyl cyclase stimulation. Therefore, cyclic AMP-dependent phosphorylation of L-type Ca2+ channels might be involved in the inotropic effect of glucagon. There have been no reports, however, of the effects of glucagon on the cardiac Ca2+ current (ICa). Also, the physiological effects of glucagon could involve mechanisms other than stimulation of adenylyl cyclase. Here we show that glucagon enhances ICa in frog and rat ventricular myocytes. The effect of glucagon in rats resulted from a stimulation of adenylyl cyclase. In frogs, however, the effect of glucagon on ICa was smaller and occurred at a concentration tenfold lower than in rats, and adenylyl cyclase was not modified. In addition, cAMP potentiated the effect of glucagon on ICa in frog ventricle, which correlated with the observed inhibition by glucagon of low-Km cAMP phosphodiesterase activity. Therefore, this is an example of a hormone that affects cardiac function in a similar way to a variety of synthetic cardiotonic compounds, such as milrinone and Ro-20-1724. Inhibition of phosphodiesterase activity by glucagon may be essential in animals in which glucagon increases cardiac contractility but does not effectively stimulate adenylyl cyclase.     

Sympathetic regulation of cardiac calcium current is due exclusively to cAMP-dependent phosphorylation.

The positive inotropic effect of the sympathetic nervous system on the heart is partly mediated by an increase in the voltage-gated Ca2+ current (ICa). This increase is generally attributed to beta-adrenergic receptor-stimulated cyclic AMP-dependent phosphorylation of the Ca2+ channel. It has been suggested that cAMP-dependent phosphorylation cannot explain all the effects of beta-adrenergic agonists on ICa and that a parallel membrane-delimited pathway involving the 'direct' action of the G protein Gs also stimulates ICa. A precedent exists for such a membrane-delimited pathway in the activation of a K+ channel by acetylcholine in heart. A membrane-delimited pathway for stimulation of ICa might be important in rapid beat-to-beat regulation of contraction by the sympathetic nervous system, because isoproterenol may produce a biphasic increase in ICa with the rapid phase (tau = 150 ms) putatively mediated by the direct pathway and the slow phase (tau = 35 s) by cAMP-dependent phosphorylation. Here we report that in frog, rat, and guinea pig ventricular myocytes ICa increases slowly and monophasically in response to isoproterenol. The increase is completely blocked by inhibitors of cAMP-dependent phosphorylation. Furthermore, the time course of the increase in ICa closely parallels the increase in contractile force produced by sympathetic nerve stimulation. These data refute earlier suggestions that regulation of Ca2+ channels by the sympathetic nervous system involves or requires a direct G-protein pathway.     

Arachidonic acid metabolites as intracellular modulators of the G protein-gated cardiac K+ channel

Arachidonic acid is released from cell membranes in response to receptor-dependent as well as receptor-independent stimulation in various cells, including cardiac myocytes. Arachidonic acid is converted to prostaglandins by cyclooxygenase and to leukotrienes by 5-lipoxygenase, metabolites which are very biologically active and modulate cellular functions such as platelet aggregation, smooth muscle contraction and neural excitation. The molecular mechanisms underlying their modulations are, however, still badly understood. Here, we report that the 5-lipoxygenase metabolites of arachidonic acid activate the pertussis toxin-sensitive G protein-gated muscarinic K+ channel (IK.ACh): arachidonic acid activation of IK.ACh was prevented by the lipoxygenase inhibitors, nordihydroguaiaretic acid and AA-861; leukotriene A4 and C4 activated IK.ACh. The activation occurred in pertussis toxin-treated atrial cells and ceased when inside-out patches were formed but the patches were still susceptible to stimulation by GTP and to inhibition by GDP-beta-S. These results indicate that arachidonic acid metabolites may stimulate the G-protein in a receptor-independent way.     

ATP对胞内钙信号的调控机制研究

以大鼠胰腺β细胞和INS-1β细胞系为研究对象,采用显微荧光测钙技术,研究了胞外ATP对胞内Ca^2＋信号的影响,初步探讨了其作用机制．实验表明：胞外ATP能够分别使大鼠胰腺β细胞和INS-1细胞内的游离Ca^2＋浓度显著升高,但2种细胞的钙信号来源不同．在大鼠胰腺β细胞中,胞外ATP主要通过动员胞内钙库释放而引起胞浆内Ca^2＋浓度显著增高;而在INS-1细胞内,胞外ATP主要通过引起胞外Ca^2＋内流而引起胞浆内Ca^2＋浓度增加．     

cGMP-dependent protein kinase enhances Ca2+ current and potentiates the serotonin-induced Ca2+ current increase in snail neurones

Protein phosphorylation catalysed by cyclic AMP-dependent, Ca2+/calmodulin-dependent and Ca2+/diacylglycerol-dependent protein kinases is important both in the modulation of synaptic transmission and in the regulation of neuronal membrane permeability (for reviews see refs 5-7). However, there has previously been no evidence for the involvement of cyclic GMP-dependent protein kinase (cGMP-PK) in the regulation of neuronal function. Serotonin induces an increase of Ca2+ current in a group of identified ventral neurones of the snail Helix aspersa. This effect is probably mediated by cGMP because it is mimicked by the intracellular injection of cGMP or the application of zaprinast, an inhibitor of cGMP-dependent phosphodiesterase. We have now found that the effect of either serotonin or zaprinast on the Ca2+ current is potentiated by the intracellular injection of cGMP-PK. Moreover, the intracellular injection of activated cGMP-PK (cGMP-PK + 1 microM cGMP) greatly enhances the Ca2+ current of the identified ventral neurones seen in the absence of serotonin. These results indicate that cGMP-PK has a physiological role in the control of the membrane permeability of these neurones.     

Direct activation of cardiac pacemaker channels by intracellular cyclic AMP.

Cyclic AMP acts as a second messenger in the modulation of several ion channels that are typically controlled by a phosphorylation process. In cardiac pacemaker cells, adrenaline and acetylcholine regulate the hyperpolarization-activated current (if), but in opposite ways; this current is involved in the generation and modulation of pacemaker activity. These actions are mediated by cAMP and underlie control of spontaneous rate by neurotransmitters. Whether the cAMP modulation of if is mediated by channel phosphorylation is, however, still unknown. Here we investigate the action of cAMP on if in excised patches of cardiac pacemaker cells and find that cAMP activates if by a mechanism independent of phosphorylation, involving a direct interaction with the channels at their cytoplasmic side. Cyclic AMP activates if by shifting its activation curve to more positive voltages, in agreement with whole-cell results. This is the first evidence of an ion channel whose gating is dually regulated by voltage and direct cAMP binding.     

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.     

铝离子对低温好氧颗粒污泥颗粒化的强化作用

研究了低温条件下Al 3+对好氧颗粒污泥颗粒化的强化作用.在Al 3+质量浓度为30.0mg.L-1条件下,经强化颗粒化作用的好氧颗粒污泥仅需40d即可以培养成熟,其表面光滑,结构紧密,具有较好的沉淀性能和较高的生物量.培养成熟的低温好氧颗粒污泥对污水具有较高的处理效能,COD,NH4+-N和PO34--P去除率分别达到85.6%,88.8%和91.9%,较好地实现了碳氮磷的低温高效同步去除.低温好氧颗粒污泥形成过程中,胞外聚合物中蛋白质类的质量分数明显升高,达到9.25mg.g-1,蛋白质类与多糖类的质量比(PN/PS)为1.10,说明较高含量的蛋白质类是好氧颗粒污泥形成的重要因素.同时,加入30mg.L-1 Al 3+后,污泥zeta电位从-18.40mV逐渐升高到-6.51mV,表明污泥之间静电斥力减小有利于污泥聚集,形成颗粒.     

细胞内Ca~(2+)对可释放囊泡库的影响

目的:探讨细胞内Ca~(2+)对可释放囊泡库的影响.方法:取体外培养成熟的新生鼠大脑皮层神经元细胞,分别对正常细胞、去除细胞外Ca~(2+)的细胞、同时去除细胞内外Ca~(2+)的细胞,记录微小型兴奋性突触后电流(mEPSCs)和微小型抑制性突触后电流(mIPSCs),观察兴奋性和抑制性突触可释放囊泡库(RRP)大小,研究细胞外和细胞内Ca~(2+)对细胞胞吐的不同影响.结果:去除神经元细胞外Ca~(2+)和细胞内Ca~(2+)均对mEPSCs和mIPSCs的电流频率有抑制作用,细胞外Ca~(2+)对RRP大小并无显著影响,而去除细胞内Ca~(2+)可引起细胞内RRP的显著降低.结论:细胞内Ca~(2+)可影响RRP的维持.     

Turning of nerve growth cones induced by localized increases in intracellular calcium ions

Guidance of developing axons involves turning of the motile tip, the growth cone, in response to a variety of extracellular cues. Little is known about the intracellular mechanism by which the directional signal is transduced. Ca2+ is a key second messenger in growth cone extension and has been implicated in growth-cone turning. Here I report that a direct, spatially restricted elevation of intracellular Ca2+ concentration ([Ca2+]i) on one side of the growth cone by focal laser-induced photolysis (FLIP) of caged Ca2+ consistently induced turning of the growth cone to the side with elevated [Ca2+]i (attraction). Furthermore, when the resting [Ca2+]i at the growth cone was decreased by the removal of extracellular Ca2+, the same focal elevation of [Ca2+]i by FLIP induced repulsion. These results provide direct evidence that a localized Ca2+ signal in the growth cone can provide the intracellular directional cue for extension and is sufficient to initiate both attraction and repulsion. By integrating local and global Ca2+ signals, a growth cone could thus generate different turning responses under different environmental conditions during guidance.