Measurement of the intracellular free calcium concentration in salamander rods

Measurement of the free calcium concentration within a photo-receptor outer segment has been considered an important aim since the proposal by Hagins and Yoshikami that the primary event in phototransduction is a release of Ca (2+) inside the cell. More recent evidence has cast doubt on the calcium hypothesis, and the observations of Yau and Nakatani and Matthews et al. suggest that the internal Ca (2+) concentration ([Ca (2+)]i), may decrease after a flash of light. In the present study we have measured [Ca (2+)]i directly by using a new method for incorporating the Ca-sensitive photoprotein aequorin into an isolated rod. We report that the light response is accompanied by a decrease in [Ca (2+)]i, caused by the closure of light-sensitive channels which are the main route for Ca (2+) entry into the outer segment. Of the Ca (2+) entering through light-sensitive channels, about 95% is sequestered by a rapid and reversible buffering mechanism. Calcium is removed from the cell by an electrogenic pump in which 3 Na (+) ions are exchanged for each Ca (2+); the pump is highly active and the free Ca (2+) in the cell declines with a time constant of ~0.5 s after a flash of light.     

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.     

Light-suppressible, cyclic GMP-sensitive conductance in the plasma membrane of a truncated rod outer segment

Recent experiments by Fesenko et al and ourselves have shown that excised membrane patches from retinal rod outer segments contain a cyclic GMP-sensitive conductance which has electrical properties similar to those of the light-sensitive conductance. This finding supports the notion that cGMP mediates phototransduction (see ref. 3) by directly modulating the light-sensitive conductance. However, some uncertainty remained about whether the patch experiments had discriminated completely between plasma and intracellular disk membranes; thus the cGMP response in an excised membrane could have resulted from contaminating disk membrane fragments, which are known to contain a cGMP-regulated conductance. Furthermore, the patch conductance has not yet been shown to be light-suppressible, an ultimate criterion for identity with the light-sensitive conductance. We now report experiments on a truncated rod outer segment preparation which resolved these issues. The results demonstrated that the cGMP-sensitive conductance was present in the plasma membrane of the outer segment, and that in the presence of GTP the conductance could be suppressed by a light flash. With added ATP, the effectiveness of the light flash was reduced and the suppression was more transient. The effects of both GTP and ATP were consistent with the known biochemistry. From the maximum current inducible by cGMP, we estimate that approximately 1% of the light-sensitive conductance is normally open in the dark; this would give an effective free cGMP concentration of a few micromolar in the intact outer segment in the dark.     

Effects on the photoresponse of calcium buffers and cyclic GMP incorporated into the cytoplasm of retinal rods

It is generally accepted that the light response in retinal rods involves a reduction of ionic permeability (predominantly to Na+) in the plasma membrane of the outer segment and that this is mediated by an internal messenger which diffuses between the disk and plasma membranes. There is controversy, however, over the identity of the diffusible substance; two alternative schemes have received widespread support (for review see refs 1,2). According to the 'calcium hypothesis', light stimulates the release into the cytoplasm of calcium, leading to the blockage of channels which are normally open in darkness, whereas based on the 'cyclic nucleotide hypothesis', cyclic GMP causes the opening of channels in the dark, but is hydrolysed by a light-activated phosphodiesterase. We report here effects of introducing calcium buffers and cyclic GMP into the rod cytoplasm by means of a patch pipette, which seem to be inconsistent with the calcium hypothesis.     

Cyclic GMP can increase rod outer-segment light-sensitive current 10-fold without delay of excitation

To test the hypothesis that cyclic GMP is the internal messenger coupling rhodopsin activation to membrane excitation in vertebrate rod photoreceptors, we used a novel technique combining measurement of membrane currents of isolated salamander rods with a suction electrode and the introduction of cyclic GMP through a whole-cell recording patch pipette. Rupture of an attached patch was followed by a rapid (approximately 10 s), approximately 10-fold increase in outer-segment membrane current, all of which was light-sensitive. There was little change in the rising phase of the response to a saturating flash, but the duration of the saturated phase of the response increased approximately 10-fold. The effects reversed completely within 3-4 min after withdrawal of the cyclic GMP-containing patch pipette. A formal kinetic analysis shows that the first two observations are inconsistent with the postulate that cyclic GMP opens the light-sensitive conductance by simple binding to channels, unless free cyclic GMP in the outer segment is assumed to be much lower than published estimates, and most of the outer-segment cyclic GMP is bound and inexchangeable on the timescale of 200 ms. Furthermore, our results suggest that rod cyclic GMP is not involved solely in keeping the light-sensitive conductance open, but may also affect the activity of the phosphodiesterase that mediates cyclic GMP hydrolysis.     

Induction by cyclic GMP of cationic conductance in plasma membrane of retinal rod outer segment

Vertebrate rod photoreceptors hyperpolarize when illuminated, due to the closing of cation-selective channels in the plasma membrane. The mechanism controlling the opening and closing of these channels is still unclear, however. Both 3',5'-cyclic GMP and Ca2+ ions have been proposed as intracellular messengers for coupling the light activation of the photopigment rhodopsin to channel activity and thus modulating light-sensitive conductance. We have now studied the effects of possible conductance modulators on excised 'inside-out' patches from the plasma membrane of the rod outer segment (ROS), and have found that cyclic GMP acting from the inner side of the membrane markedly increases the cationic conductance of such patches (EC50 30 microM cyclic GMP) in a reversible manner, while Ca2+ is ineffective. The cyclic GMP-induced conductance increase occurs in the absence of nucleoside triphosphates and, hence, is not mediated by protein phosphorylation, but seems rather to result from a direct action of cyclic GMP on the membrane. The effect of cyclic GMP is highly specific; cyclic AMP and 2',3'-cyclic GMP are completely ineffective when applied in millimolar concentrations. We were unable to recognize discrete current steps that might represent single-channel openings and closings modulated by cyclic GMP. Analysis of membrane current noise shows the elementary event to be 3 fA with 110 mM Na+ on both sides of the membrane at a membrane potential of -30 mV. If the initial event is assumed to be the closure of a single cyclic GMP-sensitive channel, this value corresponds to a single-channel conductance of 100 fS. It seems probable that the cyclic GMP-sensitive conductance is responsible for the generation of the rod photoresponse in vivo.     

Activation of a calcium-dependent photoprotein by chemical signalling through gap junctions

In the hydrozoan coelenterate Obelia geniculata, epithelial cell action potentials trigger light emission from photocyte effector cells containing obelin, an endogenous calcium-activated photoprotein. As this luminescence is blocked by the removal of extracellular calcium it seemed likely that calcium entry via voltage-gated channels in the photocyte membrane would account for the light emission. However, no inward calcium current was detected in whole cell recordings from dissociated photocytes and depolarization of isolated photocytes produced no luminescence. In contrast, a voltage-dependent calcium current was recorded from non-luminescent support cells, and activation of this current triggered luminescence in an adjacent photocyte. Surprisingly, light emission was abolished when the gap junctions between the photocyte and support cell were blocked. We conclude that calcium entry into support cells leads to light emission from neighbouring photocytes via chemical signalling through intercellular gap junctions.     

Highly cooperative feedback control of retinal rod guanylate cyclase by calcium ions

Visual excitation in retinal rod cells is mediated by a cascade that leads to the amplified hydrolysis of cyclic GMP (cGMP) and the consequent closure of cGMP-activated cation-specific channels in the plasma membrane. Recovery of the dark state requires the resynthesis of cGMP, which is catalysed by guanylate cyclase, an axoneme-associated enzyme. The lowering of the cytosolic calcium concentration (Cai) following illumination is thought to be important in stimulating cyclase activity. This hypothesis is supported by the finding that the cGMP content of rod outer segments increases several-fold when Cai is lowered to less than 10 nM. It is evident that cGMP and Cai levels are reciprocally controlled by negative feedback. Guanylate cyclase from toad ROS is strongly stimulated when the calcium level is lowered from 10 microM to 10 nM, but only if they are excited by light. We show here that the guanylate cyclase activity of unilluminated bovine rod outer segments increases markedly (5 to 20-fold) when the calcium level is lowered from 200 nM to 50 nM. This steep dependence of guanylate cyclase activity on the calcium level in the physiological range has a Hill coefficient of 3.9. Stimulation at low calcium levels is mediated by a protein that can be released from the outer segment membranes by washing with a low salt buffer. Calcium sensitivity is partially restored by adding the soluble extract back to the washed membranes. The highly cooperative activation of guanylate cyclase by the light-induced lowering of Cai is likely to be a key event in restoring the dark current after excitation.     

Electrogenic Na-Ca exchange in retinal rod outer segment

Previous work has suggested that a Na-Ca exchanger may have a key role in visual transduction in retinal rods. This exchanger is thought to maintain a low internal free Ca2+ concentration in darkness and to contribute to the rod's recovery after light by removing any internally released Ca2+. Little else is known about this transport mechanism in rods. We describe here an inward membrane current recorded from single isolated rods which appears to be associated with such external Na+-dependent Ca2+ efflux activity. External Na+, but not Li+, could generate this current; high external K+ inhibited it while small amounts of La3+ (10 microM) completely abolished it. The exchanger can also transport Sr2+, but not Ba2+ or other divalent cations. The exchange ratio was estimated to be 3Na+:1Ca2+. As well as demonstrating clearly the Na-Ca exchanger in the rod outer segment, our experiments also cast serious doubt on the commonly held view that light simply releases internal Ca2+ to bind to and block the light-sensitive conductance.     

Mechanism of ion permeation through calcium channels

Calcium channels carry out vital functions in a wide variety of excitable cells but they also face special challenges. In the medium outside the channel, Ca2+ ions are vastly outnumbered by other ions. Thus, the calcium channel must be extremely selective if it is to allow Ca2+ influx rather than a general cation influx. In fact, calcium channels show a much greater selectivity for Ca2+ than sodium channels do for Na+ despite the high flux that open Ca channels can support. Relatively little is known about the mechanism of ion permeation through Ca channels. Earlier models assumed ion independence or single-ion occupancy. Here we present evidence for a novel hypothesis of ion movement through Ca channels, based on measurements of Ca channel activity at the level of single cells or single channels. Our results indicate that under physiological conditions, the channel is occupied almost continually by one or more Ca2+ ions which, by electrostatic repulsion, guard the channel against permeation by other ions. On the other hand, repulsion between Ca2+ ions allows high throughput rates and tends to prevent saturation with calcium.     

春小麦感光性遗传的初步研究

小麦是数量长日植物,其不同品种对日长反应的敏感程度不同。利用在广东冬季补充长光照条件下比自然短光照条件下的提前抽穗促进率,可以间接表示不同杂交组合的感光性强弱。用感光性强弱不等的春小麦亲本配置出的杂交组合,其子代与亲代,下代与上代的感光性之间存在着密切相关。如果要选育广适性的感光性弱的品种,最好双亲都选用感光性弱的材料,至少亲本之一的感光性要弱。在短光照条件下,杂种后代感光性不同的类型明显分离,抽穗天数遗传力较高。在F_2代根据抽穗早晚对感光性进行选择就可以得到较可靠的效果。因此,利用在广东冬繁时的短光照条件,可以选择出感光性弱而适应性广的春小麦材料。     

Cyclic GMP-sensitive conductance in outer segment membrane of catfish cones

A cyclic GMP-sensitive conductance has recently been observed with patch-clamp recording in excised inside-out patches of plasma membrane from frog and toad rod outer segments. This conductance has properties suggesting that it is probably the light-sensitive conductance involved in visual transduction. We now report a similar conductance in the outer segment membrane of catfish cones. Cyclic GMP showed positive cooperativity in opening this conductance, with a Hill coefficient of 1.6-3.0 and a half-saturating cGMP concentration of 35-70 microM. Cyclic AMP at 1 mM, or changing Ca concentration (in the presence of Mg), had little effect on the conductance. In physiological solutions the cGMP-induced current had a reversal potential near +10 mV; the current amplitude increased roughly exponentially with membrane potential in both depolarizing and hyperpolarizing directions. Our results suggest that cGMP is also the internal transmitter for phototransduction in cones.     

Augmentation of cardiac calcium current by flash photolysis of intracellular caged-Ca2+ molecules

The entry of calcium ions into cells through voltage-activated Ca2+ channels in the plasma membrane triggers many important cellular processes. The activity of these channels is regulated by several hormones and neurotransmitters, as well as intracellular messengers such as Ca2+ itself (for examples, see refs 1-9). In cardiac muscle, myoplasmic Ca2+ has been proposed to potentiate Ca2+ influx, although a direct effect of Ca2+ on these channels has not yet been demonstrated. Photosensitive 'caged-Ca2+' molecules such as nitr-5, however, provide powerful tools for investigating possible regulatory roles of Ca2+ on the functioning of Ca2+ channels. Because its affinity for Ca2+ is reduced by irradiation, nitr-5 can be loaded into cells and induced to release Ca2+ with a flash of light. By using this technique we found that the elevation of intracellular Ca2+ concentration directly augmented Ca2+-channel currents in isolated cardiac muscle cells from both frog and guinea pig. The time course of the current potentiation was similar to that seen with beta-adrenergic stimulation. Thus Ca2+ may work through a similar pathway, involving phosphorylation of a regulatory Ca2+-channel protein. This mechanism is probably important for the accumulation of Ca2+ and the amplification of the contractile response in cardiac muscle, and may have a role in other excitable cells.     

Cloning of a probable potassium channel gene from mouse brain

Potassium channels comprise a diverse class of ion channels important for neuronal excitability and plasticity. The recent cloning of the Shaker locus from Drosophila melanogaster has provided a starting point for molecular studies of potassium channels. Predicted Shaker proteins appear to be integral membrane proteins and have a sequence similar to the sequence of the S4 segment of the vertebrate sodium channel, where the S4 segment has been proposed to be the voltage sensor. Expression studies in frog oocytes confirm that Shaker encodes a component of a potassium channel (the A channel) that conducts a fast transient potassium current. Here we report the isolation of complementary DNA clones from the mouse brain, the nucleotide sequences of which predict a protein remarkably similar to the Shaker protein. The strong conservation of the predicted protein sequence in flies and mammals suggests that these mouse clones encode a potassium channel component and that the conserved amino acids may be essential to some aspect of potassium channel function.     

6种化学消毒剂对养殖海水中浮游生物的影响

 采用不同质量浓度的聚维酮碘、二溴海因、福尔马林、富氯、漂粉精和次氯酸钠等6种化学消毒剂,对蓄水沉淀池养殖海水进行消毒处理,处理前后分别测定浮游生物种类和数量变化。结果显示：在合理的浓度范围内,各种化学消毒剂对浮游生物均有一定程度的影响,和质量浓度成正相关变化。含氯消毒剂作用效果最好,有效氯达到10 g·m^-3时,漂粉精对浮游动物杀灭率最高,达到97.3%,次氯酸钠次之,为91.4%。本文研究结果对对虾养殖用水的处理和循环利用有重要的指导意义,有利于部分切断对虾白斑综合症病毒的水平传播途径,减少病害的发生,促进对虾养殖业的健康发展。     

心房纤维性颤动电生理重构机制研究进展

心房纤维性颤动(AF)是临床上常见的持续性快速心律失常,近年研究显示其具有自身延续性,即有心房电生理重构,对其发病机理有新的理解。形成心房电生理重构的主要机制是离子通道的重构。其中,在影响心房电生理重构的过程中,不同的钠离子通道、钾离子通道和钙离子通道均表现出重构现象。而mRNA浓度下降会导致通道电流密度降低。离子通道的变化既是AF的结果,又是维持AF的电生理基础。许多抑制AF的药物作用机理是减少电生理重构,故AF电生理重构的机制是十分复杂的问题。     

Mechanism of calcium channel blockade by verapamil, D600, diltiazem and nitrendipine in single dialysed heart cells

Organic inhibitors of calcium influx prevent outward as well as inward current through cardiac calcium channels but do not slow current activation. Although block is antagonized by raising external calcium or barium concentrations, the competitive effect of permeant cations does not occur at the same cation binding site at which inorganic blockers act. Organic drugs show varying degrees of use-dependent block, due in part to blockade of open channels. Nitrendipine blockade of calcium currents requires doses greater than 100-fold higher than expected from radioligand binding to isolated membranes.     

The voltage-sensitive sodium channel is a bell-shaped molecule with several cavities

Voltage-sensitive membrane channels, the sodium channel, the potassium channel and the calcium channel operate together to amplify, transmit and generate electric pulses in higher forms of life. Sodium and calcium channels are involved in cell excitation, neuronal transmission, muscle contraction and many functions that relate directly to human diseases. Sodium channels--glycosylated proteins with a relative molecular mass of about 300,000 (ref. 5)--are responsible for signal transduction and amplification, and are chief targets of anaesthetic drugs and neurotoxins. Here we present the three-dimensional structure of the voltage-sensitive sodium channel from the eel Electrophorus electricus. The 19 A structure was determined by helium-cooled cryo-electron microscopy and single-particle image analysis of the solubilized sodium channel. The channel has a bell-shaped outer surface of 135 A in height and 100 A in side length at the square-shaped bottom, and a spherical top with a diameter of 65 A. Several inner cavities are connected to four small holes and eight orifices close to the extracellular and cytoplasmic membrane surfaces. Homologous voltage-sensitive calcium and tetrameric potassium channels, which regulate secretory processes and the membrane potential, may possess a related structure.     

NMDA spikes in basal dendrites of cortical pyramidal neurons

Basal dendrites are a major target for synaptic inputs innervating cortical pyramidal neurons. At present little is known about signal processing in these fine dendrites. Here we show that coactivation of clustered neighbouring basal inputs initiated local dendritic spikes, which resulted in a 5.9 +/- 1.5 mV (peak) and 64.4 +/- 19.8 ms (half-width) cable-filtered voltage change at the soma that amplified the somatic voltage response by 226 +/- 46%. These spikes were accompanied by large calcium transients restricted to the activated dendritic segment. In contrast to conventional sodium or calcium spikes, these spikes were mediated mostly by NMDA (N-methyl-D-aspartate) receptor channels, which contributed at least 80% of the total charge. The ionic mechanism of these NMDA spikes may allow 'dynamic spike-initiation zones', set by the spatial distribution of glutamate pre-bound to NMDA receptors, which in turn would depend on recent and ongoing activity in the cortical network. In addition, NMDA spikes may serve as a powerful mechanism for modification of the cortical network by inducing long-term strengthening of co-activated neighbouring inputs.     

鸡骨香乙醇提取物舒张小鼠气道平滑肌的作用机理研究

为研究鸡骨香乙醇提取物(EECC)舒张小鼠气道平滑肌的作用机理.在组织水平上,利用生物机能系统,检测EECC对气管张力的影响.在细胞水平上,利用膜片钳系统,记录EECC对L型电压依赖性钙通道(VDLCCs)电流的影响.在活体水平上,利用肺功能仪,检测EECC对小鼠呼吸系统阻力(Rrs)的影响.实验发现在组织水平上,EE...