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.     

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.     

Effect of ions on the light-sensitive current in retinal rods

The effect of ions on the light-sensitive current of retinal rods was studied by sucking the inner segment into a tightly fitting capillary with the outer segment projecting into a flowing solution. This new method showed that the light-sensitive pathway, in which Na+ is the normal carrier of current, has an ionic selectivity different from that of other known sodium channels. Externàl calcium has a striking effect on the current, which increased about 20-fold when all calcium was removed. Reducing the sodium concentration gradient greatly prolonged the response to a flash of light, as would be expected if internal calcium blocks sodium channels and if light releases calcium which is subsequently extruded by a sodium-calcium exchange mechanism.     

Opposite effects of cyclic GMP and cyclic AMP on Ca2+ current in single heart cells

The slow inward Ca2+ current, ICa, is fundamental in the initiation of cardiac contraction and neurohormonal regulation of cardiac function. It is increased by beta-adrenergic agonists, which stimulate synthesis of cyclic AMP (cAMP) and cAMP-dependent phosphorylation. The neurotransmitter acetylcholine reduces ICa by an unknown mechanism. There is strong evidence that acetylcholine reduces ICa by decreasing adenylate cyclase activity, but cGMP has also been implicated as ACh stimulates cGMP accumulation and activates cGMP-dependent protein kinase. Application of cGMP decreases contractile force, decreases Ca flux, shortens the duration of action potentials and inhibits Ca-dependent action potentials. Other studies, however, have concluded that cGMP levels do not correlate with contractile force and that cGMP has no effect on ICa. We have therefore examined the effects of intracellular perfusion of cGMP on ICa using isolated, voltage-clamped cells from frog ventricle. We find that cGMP has negligible effects on basal ICa, but greatly decreases the ICa that had been elevated by beta-adrenergic agonists or by intracellular perfusion with cAMP. The decrease of ICa is mediated by cAMP hydrolysis via a cGMP-stimulated cyclic nucleotide phosphodiesterase.     

Cyclic GMP increases photocurrent and light sensitivity of retinal cones

Like retinal rods, cone photoreceptors contain cyclic GMP and light-activated phosphodiesterase. The cGMP phosphodiesterase cascade is thought to mediate phototransduction in rods. Biochemical assays of nucleotide content in cone-dominant retinas, however, have failed to demonstrate light-induced changes in cGMP. Changes in cyclic AMP following light exposure have been reported, leading to the suggestion that in cone phototransduction cAMP assumes a role analogous to that played by cGMP in rods. Cyclic GMP introduced from tight-seal pipettes into isolated cones of the larval tiger salamander, Ambystoma tigrinum, rapidly increases light-modulated membrane current more than 10-fold. In cones, as in rods, cGMP also causes an approximately 10-fold increase in photocurrent duration and a 5- to 10-fold increase in light-sensitivity. Cyclic AMP has no effect on cone photocurrents under the same conditions. Because cGMP has similar effects on photocurrent magnitude and kinetics in both rods and cones, we conclude that cGMP plays corresponding roles in transduction in both vertebrate photoreceptor classes.     

本征晶格缺陷对BNT铁电薄膜光响应性能的影响简

用溶胶-凝胶法在YSZ/Si衬底上制备Bi3．15 Nd0．85 Ti3 O12（BNT）铁电薄膜,研究了退火气氛和退火温度对BNT薄膜的光响应性能的影响。对不同退火气氛和退火温度下的BNT薄膜进行微观结构和光响应性能表征。研究结果表明：随着退火气氛中氧含量的降低,光响应增大,BNT薄膜中氧空位起到了为光生载流子传输提供通道的作用;随着退火温度的降低,光开启电压和饱和光电导增大,BNT薄膜中高密度的晶界虽然阻碍了光生载流子的迁移,却有利于使光生载流子在晶界处及时分开。     

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.     

基于代建制模式的GMP机制设计探讨

介绍美国工程项目管理中CM@R模式GMP（guaranteed maximum price）控制投资的过程。用委托代理理论解析GMP投资控制设计机制、比较CM@R与代建制投资控制设计机制差异,提出代建制模式下应用GMP控制投资的机制设计建议,可分为三个阶段应用实施：两阶段招标法;联合投标选择代建单位;代建单位总包。     

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.