scholarly journals Effects of cyclic GMP on the kinetics of the photocurrent in rods and in detached rod outer segments.

1987 ◽  
Vol 90 (4) ◽  
pp. 527-551 ◽  
Author(s):  
S Hestrin ◽  
J I Korenbrot
Keyword(s):  
Cyclic Gmp ◽  
Outer Segment ◽  
Time Course ◽  
Intact Cell ◽  
Light Sensitivity ◽  
Biochemical Properties ◽  
Intact Cells ◽  
Outer Segments ◽  
High Concentrations ◽  
Kinetics Of

We investigated the effects of high concentrations of cytoplasmic cyclic GMP on the photocurrent kinetics and light sensitivity of the tiger salamander rod both in intact cells and in detached outer segments. Photoreceptors were internally perfused with cGMP by applying patch pipettes containing cGMP to the inner or outer segment. Large increases in the concentration of cGMP in the outer segment cytoplasm were achieved only when the patch pipette was applied directly to the outer segment. The dark-current amplitude increased with increasing cGMP concentrations up to approximately 1,400 pA. Internal perfusion with 5.0 mM cGMP introduced a delay of 1-3 s in the photocurrent. The magnitude of the delay was inversely proportional to the light intensity. In addition, the photocurrent time course was slowed down and the light sensitivity, measured 1 s after the flash, was decreased approximately 100-fold when compared with that of the intact cell. The observed effects of cGMP were compared with those predicted by a model that assumes that the initial photocurrent time course is determined by the kinetics of the light-activated phosphodiesterase (PDE) and the cGMP dependence of the light-sensitive channels. At high concentrations of cGMP, the experimental data were similar to those predicted by the model and based on the known biochemical properties of the light-activated PDE and cGMP-activated channels.

scholarly journals Control of the cyclic GMP phosphodiesterase of frog photoreceptor membranes.

1980 ◽  
Vol 76 (5) ◽  
pp. 631-645 ◽  
Author(s):  
P R Robinson ◽  
S Kawamura ◽  
B Abramson ◽  
M D Bownds
Keyword(s):  
Light Intensity ◽  
Cyclic Gmp ◽  
Outer Segment ◽  
Calcium Concentration ◽  
Light Adaptation ◽  
Light Sensitivity ◽  
Change Mechanism ◽  
Outer Segments ◽  
Labile Component ◽  
Photoreceptor Membranes

The light-activated cyclic GMP phosphodiesterase (PDE) of frog photoreceptor membranes has been assayed in isolated outer segments suspended in a low-calcium Ringer's solution. Activation occurs over a range of light intensity that also causes a decrease in the permeability, cyclic GMP levels, and GTP levels of isolated outer segments. At intermediate intensities, PDE activity assumes constant intermediate values determined by the rate of rhodopsin bleaching. Washing causes an increase in maximal enzyme activity. Increasing light intensity from darkness to a level bleaching 5 x 10(3) rhodopsin molecules per outer segment per second shifts the apparent Michaelis constant (Km) from 100 to 900 microM. Maximum enzyme velocity increases at least 10-fold. The component that normally regulates this light-induced increase in the Km of PDE is removed by the customary sucrose flotation procedures. The presence of 10(-3) M Ca++ increases the light sensitivity of PDE, and maximal activation is caused by illumination bleaching only 5 x 10(2) rhodopsin molecules per outer segment per second. Calcium acts by increasing enzyme velocity while having little influence on Km. The effect of calcium appears to require a labile component, sensitive to aging of the outer segment preparation. The decrease in the light sensitivity of PDE that can be observed upon lowering the calcium concentration may be related to the desensitization of the permeability change mechanism that occurs during light adaptation of rod photoreceptors.

scholarly journals Kinetics of light-dependent Ca fluxes across the plasma membrane of rod outer segments. A dynamic model of the regulation of the cytoplasmic Ca concentration.

1987 ◽  
Vol 90 (3) ◽  
pp. 397-425 ◽  
Author(s):  
D L Miller ◽  
J I Korenbrot
Keyword(s):  
Plasma Membrane ◽  
Outer Segment ◽  
Time Course ◽  
Small Volume ◽  
Dynamic Balance ◽  
Light Sensitivity ◽  
Ca Ions ◽  
Concentration Changes ◽  
The Difference ◽  
Kinetics Of

We measured simultaneously in single toad rods the membrane photocurrent and the Ca concentration in a small volume surrounding the outer segment. Illumination causes a rise in the extracellular Ca concentration. Photocurrents and Ca concentration changes occur over the same range of light intensities. Analysis of the time course of the Ca concentration changes suggests that these concentration changes arise from the difference in the transport rates of light-activated Ca influx and efflux across the outer segment plasma membrane. The Ca influx occurs through the light-sensitive channels of the outer segment membrane and the efflux through Na/Ca exchangers. In 0.1 mM external Ca, approximately 1-2% of the dark current is carried by Ca ions. The Ca efflux in the dark is identical to the influx, approximately 2 X 10(6) ions/s. Upon illumination, the Ca influx decreases with a time course and light sensitivity identical to those of the photocurrent. The Ca efflux, on the other hand, has very different kinetics from those of the photocurrent. Upon illumination, the Ca efflux decreases with a time course and light sensitivity determined by the change in membrane voltage and in the free cytoplasmic Ca concentration near the plasma membrane. In response to bright stimuli, which saturate the photocurrent for prolonged periods of time, the Ca efflux decays with an exponential time course from its value in darkness. The average time constant of this decay is 2.5 s. From the kinetics of the light-activated Ca fluxes, it is possible to predict that illumination causes a decrease in the cytoplasmic Ca concentration. We present a model of the regulation of the cytoplasmic Ca concentration by the dynamic balance of the Ca influx and efflux from the rod outer segment. The model accounts for our experimental observations and allows us to predict the time course and extent of the light-dependent decrease in the free cytoplasmic concentration.

Cyclic AMP has no effect on the generation, recovery, or background adaptation of light responses in functionally intact rod outer segments: With implications about the function of phosducin

2000 ◽  
Vol 17 (6) ◽  
pp. 887-892 ◽  
Author(s):  
HANA JINDROVA ◽  
PETER B. DETWILER
Keyword(s):  
Outer Segment ◽  
Light Exposure ◽  
Cell Voltage ◽  
Light Sensitivity ◽  
Rod Outer Segments ◽  
Light Responses ◽  
Outer Segments ◽  
Sequence Of Events ◽  
Background Adaptation ◽  
Dependent Processes

In retinal rods, light exposure decreases the total outer segment content of both cGMP and cAMP by about 50%. The functional role of the light-evoked change in cAMP is not known. It is postulated to trigger changes in the phosphorylation state of phosducin, a phosphoprotein that is phosphorylated in the dark by cAMP-dependent protein kinase (PKA) and dephosphorylated by basal phosphatase activity when PKA is inhibited by the light-evoked drop in cAMP. In biochemical studies, dephosphorylated phosducin binds to free βγ dimer of transducin (Tβγ) and prevents the regeneration of heterotrimeric transducin by blocking the re-association of the βγ and α subunits. Phosducin's interaction with Tβγ is blocked when it is phosphorylated on a single residue by PKA. To evaluate the effect of the light-evoked fall in cAMP, functionally intact isolated lizard rod outer segments were dialyzed in whole-cell voltage clamp with a standard internal solution and electrical light responses were recorded with and without adding cAMP to the dialysis solution. Since the total outer segment content of cAMP in darkness is ∼5 μM, internal dialysis with solution containing a much higher concentration (100 μM) of cAMP (or 8-bromo-cAMP) will overcome the effects of a light-evoked decrease in its concentration by keeping cAMP-dependent processes fully activated. Neither cyclic nucleotide had any influence on the generation, light sensitivity, recovery, or background adaptation of the flash response. These results also argue against the participation of phosducin in the sequence of events that are responsible for these aspects of rod function. This does not exclude the possibility of phosducin being involved in adaptation caused by higher light levels than used in the present study, that is, bleaching adaptation, or in light-dependent processes other than phototransduction.

scholarly journals Kinetics of oxygen consumption after a single flash of light in photoreceptors of the drone (Apis mellifera).

1982 ◽  
Vol 80 (1) ◽  
pp. 19-55 ◽  
Author(s):  
M Tsacopoulos ◽  
S Poitry
Keyword(s):  
Oxygen Consumption ◽  
Time Constant ◽  
Time Course ◽  
Sodium Pump ◽  
Fourier Transforms ◽  
Recovery Phase ◽  
Peak Amplitude ◽  
Single Flash ◽  
High Concentrations ◽  
Kinetics Of

The time course of the rate of oxygen consumption (QO2) after a single flash of light has been measured in 300-micrometers slices of drone retina at 22 degrees C. To measure delta QO2(t), the change in QO2 from its level in darkness, the transients of the partial pressure of O2 (PO2) were recorded with O2 microelectrodes simultaneously in two sites in the slice and delta QO2 was calculated by a computer using Fourier transforms. After a 40-ms flash of intense light, delta QO2, reached a peak of 40 microliters O2/g.min and then declined exponentially to the baseline with a time constant tau 1 = 4.96 +/- 0.49 s (SD, n = 10). The rising phase was characterized by a time constant tau 2 = 1.90 +/- 0.35 s (SD, n = 10). The peak amplitude of delta QO2 increased linearly with the log of the light intensity. Replacement of Na+ by choline, known to decrease greatly the light-induced transmembrane current, caused a 63% decrease of delta QO2. With these changes, however, the kinetics of delta QO2 (t) were unchanged. This suggest that the recovery phase is rate-limited by a single reaction with apparent first-order kinetics. Evidence is provided that suggests that this reaction may be the working of the sodium pump. Exposure of the retina to high concentrations of ouabain or strophanthidin (inhibitors of the sodium pump) reduced the peak amplitude of delta QO2 by approximately 80% and increased tau 1. The increase of tau 1 was an exponential function of the time of exposure to the cardioactive steroids. Hence, it seems likely that the greatest part of delta QO2 is used for the working of the pump, whose activity is the mechanism underlying the rate constant of the descending limb of delta QO2 (t).

scholarly journals Functional significance of the taper of vertebrate cone photoreceptors

2012 ◽  
Vol 139 (2) ◽  
pp. 159-187 ◽  
Author(s):  
Ferenc I. Hárosi ◽  
Iñigo Novales Flamarique
Keyword(s):  
Outer Segment ◽  
Signal To Noise Ratio ◽  
Light Flux ◽  
Light Sensitivity ◽  
Morphological Data ◽  
Outer Segments ◽  
Primary Determinant ◽  
The One ◽  
Reduced Light ◽  
Theoretical Analyses

Vertebrate photoreceptors are commonly distinguished based on the shape of their outer segments: those of cones taper, whereas the ones from rods do not. The functional advantages of cone taper, a common occurrence in vertebrate retinas, remain elusive. In this study, we investigate this topic using theoretical analyses aimed at revealing structure–function relationships in photoreceptors. Geometrical optics combined with spectrophotometric and morphological data are used to support the analyses and to test predictions. Three functions are considered for correlations between taper and functionality. The first function proposes that outer segment taper serves to compensate for self-screening of the visual pigment contained within. The second function links outer segment taper to compensation for a signal-to-noise ratio decline along the longitudinal dimension. Both functions are supported by the data: real cones taper more than required for these compensatory roles. The third function relates outer segment taper to the optical properties of the inner compartment whereby the primary determinant is the inner segment’s ability to concentrate light via its ellipsoid. In support of this idea, the rod/cone ratios of primarily diurnal animals are predicted based on a principle of equal light flux gathering between photoreceptors. In addition, ellipsoid concentration factor, a measure of ellipsoid ability to concentrate light onto the outer segment, correlates positively with outer segment taper expressed as a ratio of characteristic lengths, where critical taper is the yardstick. Depending on a light-funneling property and the presence of focusing organelles such as oil droplets, cone outer segments can be reduced in size to various degrees. We conclude that outer segment taper is but one component of a miniaturization process that reduces metabolic costs while improving signal detection. Compromise solutions in the various retinas and retinal regions occur between ellipsoid size and acuity, on the one hand, and faster response time and reduced light sensitivity, on the other.

scholarly journals Differences in calcium homeostasis between retinal rod and cone photoreceptors revealed by the effects of voltage on the cGMP-gated conductance in intact cells.

1994 ◽  
Vol 104 (5) ◽  
pp. 909-940 ◽  
Author(s):  
J L Miller ◽  
J I Korenbrot
Keyword(s):  
Guanylyl Cyclase ◽  
Outer Segment ◽  
Time Course ◽  
Reversal Potential ◽  
Outward Current ◽  
Membrane Depolarization ◽  
Maximum Enhancement ◽  
Intact Cells ◽  
Retinal Rod ◽  
Voltage Dependent

We measured currents under voltage clamp in intact retinal rod photoreceptors with tight seal electrodes in the perforated patch mode. In the dark, membrane depolarization to voltages > or = +20 mV activates a time- and voltage-dependent outward current in the outer segment. This dark voltage-activated current (DVAC) increases in amplitude with a sigmoidal time course that is voltage dependent. DVAC reaches its maximum enhancement of approximately 30% in 4-6 s at +60 mV. DVAC is entirely suppressed by light and its current-voltage curve and reversal potential are the same as those of the photocurrent. Therefore, DVAC arises from the opening in darkness of the cGMP-gated channels of the outer segment. DVAC is blocked by BAPTA loaded into the cell's cytoplasm and is enhanced by lowering extracellular Ca2+ concentration. Because the cGMP-gated channels are not directly gated by voltage and because BAPTA blocks DVAC, we suggest this signal arises from a voltage-dependent decrease in cytoplasmic Ca2+ concentration that, in turn, activates guanylyl cyclase and causes cGMP synthesis. In rods loaded with high cytoplasmic Na+, membrane depolarization in darkness to voltages > or = +20 mV inactivates the outward current in the outer segment with an exponential time course. We call this DVIC (dark, voltage-inactivated current). DVIC reflects voltage-dependent closing of the cGMP-gated channel in the dark. DVIC, too, is blocked by cytoplasmic BAPTA, and it arises from a voltage-dependent rise in cytoplasmic Ca2+ in darkness, which occurs only if cytoplasmic Na is high. We develop a quantitative model to calculate the rate and extent of the voltage-dependent change in cytoplasmic Ca2+ concentration in a normal rod. We assume that this concentration is controlled by the balance between Ca2+ influx through the cGMP-gated channels and its efflux through a Na+/Ca2+, K+ exchanger. Lowered cytoplasmic Ca2+ is linked to guanylyl cyclase activation with characteristics determined from biochemical studies. The model considers the cytoplasmic buffering of both Ca2+ and cGMP. Simulated data generated by the model fit well DVAC measured in rods and also DVAC previously measured in cones. DVAC in cones is larger in magnitude and faster in time course than that in rods. The successful fit of DVAC by the model leads us to suggest that the activity and Ca2+ dependence of the enzymes of transduction are not different in rods and cones, but the quantitative features of Ca2+ homeostasis in the outer segment of the two receptor types differ profoundly.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals THE KINETICS OF PENETRATION

1938 ◽  
Vol 22 (2) ◽  
pp. 147-163 ◽  
Author(s):  
A. G. Jacques
Keyword(s):  
Osmotic Pressure ◽  
Free Space ◽  
Partition Coefficients ◽  
Sea Water ◽  
Intact Cell ◽  
Linear Part ◽  
Osmotic Concentration ◽  
Intact Cells ◽  
Rate Of Increase ◽  
Kinetics Of

When Valonia cells are impaled on capillaries, it is in some ways equivalent to removing the comparatively inelastic cellulose wall. Under these conditions sap can migrate into a free space and it is found that on the average the rate of increase of volume of the sap is 15 times what it is in intact cells kept under comparable conditions. The rate of increase of volume is a little faster during the first few hours of the experiment, but it soon becomes approximately linear and remains so as long as the experiment is continued. The slightly faster rate at first may mean that the osmotic pressure of the sap is approaching that of the sea water (in the intact cell the sap osmotic pressure is always slightly above that of the sea water). This might result from a more rapid entrance of water than of electrolyte, as would be expected when the restriction of the cellulose wall was removed. During the linear part of the curve the osmotic concentration and the composition of the sap suffer no change, so that entrance of electrolyte must be 15 times as fast in the impaled cells as it is in the intact cells. The explanation which best accords with the facts is that in the intact cell the entrance of electrolyte tends to increase the osmotic pressure. As a consequence the protoplasm is partially dehydrated temporarily and it cannot take up more water until the cellulose wall grows so that it can enclose more volume. The dehydration of the protoplasm may have the effect of making the non-aqueous protoplasm less permeable to electrolytes by reducing the diffusion and partition coefficients on which the rate of entrance depends. In this way the cell is protected against great fluctuations in the osmotic concentration of the sap.

Time course and kinetics of proximal tubular processing of insulin

1992 ◽  
Vol 262 (5) ◽  
pp. F813-F822 ◽  
Author(s):  
S. Nielsen
Keyword(s):  
Steady State ◽  
Time Course ◽  
Accumulation Rate ◽  
Compartmental Analysis ◽  
Proximal Tubules ◽  
Hydrolysis Rate ◽  
Time Courses ◽  
High Concentrations ◽  
Kinetics Of

The present study was undertaken to determine the time courses and kinetics of the subcellular processing of 125I-insulin in isolated and in vitro perfused proximal tubules. Morphometric analysis demonstrated well-preserved ultrastructure after 90 min of perfusion. After luminal perfusion for 90 min the absorption was constant with time and reached steady state within 5 min (177 +/- 7 fg.min-1.mm-1). Also the hydrolysis rate and tubular accumulation rate were constant and averaged 84 +/- 8 and 93 +/- 10 fg.min-1.mm-1, respectively. Free 125I appeared already within 5 min of perfusion and reached steady state within 10 min. From proximal tubules perfused with 125I-insulin for 30 min and chased for 60 min, a compartmental analysis revealed two compartments; half time (t1/2) for delivery of insulin to the lysosomes was determined to be 8.5 min, and t1/2 for lysosomal degradation was 72 min. The results demonstrated that internalization by endocytic invaginations, incorporation in endocytic vacuoles, fusion with lysosomes, and hydrolysis were rapid processes and reached maximum rates within few minutes. A significant transtubular transport of insulin to the peritubular compartment was determined to be a constant rate of 11.2 +/- 0.7 fg.min-1.mm-1. Perfusion of tubules with insulin at high concentrations in the perfusate revealed that the transport was dependent on the absorbed amount and not on the perfused load, compatible with transport through the cells and not via a paracellular mechanism. The intactness of the tight junctions was supported by the following: 1) [14C]inulin leak did not increase with time and 2) enzyme-free intercellular spaces were evident after perfusion for only 5 min with microperoxidase (mol wt of 1,700). The transported 125I-insulin was trichloroacetic acid precipitable and immunoprecipitable.

scholarly journals Studies on the time course and rate-limiting steps in the activation of adenylate cyclase in rat liver by cholera toxin

1978 ◽  
Vol 173 (1) ◽  
pp. 59-64 ◽  
Author(s):  
J Fischer ◽  
T R Kohler ◽  
L G Lipson ◽  
J Flores ◽  
P A Witkum ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Rat Liver ◽  
Time Course ◽  
Intact Cell ◽  
Intact Cells ◽  
Maximal Stimulation ◽  
Rate Limiting

Cholera toxin stimulates adenylate cyclase in rat liver after intravenous injection. The stimulation follows a short latent period of 10min, and maximum stimulation was attained at 120min. Half-maximal stimulation was achieved at 35min. In contrast with this lengthy time course in the intact cell, adenylate cyclase in broken-cell preparations of rat liver in vitro were maximally stimulated by cholera toxin (in the presence of NAD+) in 20min with half-maximal stimulation in 8min. Binding of cholera toxin to cell membranes by the B subunits is followed by translocation of the A subunit into the cell or cell membrane, and separation of the A1 polypeptide chain from the A2 chain by disulphide-bond reduction, and finally activation of adenylate cyclase by the A1 chain and NAD+. As the binding of cholera toxin is rapid, two possible rate-limiting steps could be the determinants of the long time course of action. These are translocation of the A1 chain from the outside of the cell membrane to its site of action (this includes the time required for separation from the whole toxin) or the availability of NAD+ for activation. When NAD+ concentrations in rat liver were elevated 4-fold, by the administration of nicotinamide, no change in the rate of activation of adenylate cyclase by cholera toxin was observed. Thus the intracellular concentration of NAD+ is not rate-limiting and the major rate-limiting determinant in intact cells must be between the time of toxin binding to the cell membrane and the appearance of subunit A1 at the enzyme site.

scholarly journals Amplitude, kinetics, and reversibility of a light-induced decrease in guanosine 3',5'-cyclic monophosphate in frog photoreceptor membranes.

1979 ◽  
Vol 73 (5) ◽  
pp. 629-653 ◽  
Author(s):  
M L Woodruff ◽  
M D Bownds
Keyword(s):  
Cyclic Gmp ◽  
Outer Segment ◽  
Calf Serum ◽  
Absolute Number ◽  
Continuous Illumination ◽  
Cyclic Monophosphate ◽  
Outer Segments ◽  
A Value ◽  
Anti Oxidant ◽  
Photoreceptor Membranes

The concentration of guanosine 3',5'-cyclic monophosphate (cyclic GMP) has been examined in suspensions of freshly isolated frog rod outer segments using conditions which previously have been shown to maintain the ability of outer segments to perform a light-induced permeability change (presence of calf serum, anti-oxidant, and low calcium concentration). Illumination causes a rapid decrease in cyclic GMP levels which has a half-time approximately 125 ms. With light exposures that bleach less than 100 rhodopsin molecules in each rod outer segment, at least 10(4)-10(5) molecules of cyclic GMP are hydrolyzed for each rhodopsin molecule bleached. Half of the total cyclic GMP in each outer segment, approximately 2 X 10(7) molecules, is contained in the light-sensitive pool. If outer segments are exposed to continuous illumination, using intensities which bleach between 5.0 X 10(1) and 5.0 X 10(4) rhodopsin molecules/outer segment per second, cyclic GMP levels fall to a value characteristic for the intensity used. This suggests that a balance between synthesis and degradation of cyclic GMP is established. This constant level appears to be regulated by the rate of bleaching rhodopsin molecules (by the intensity of illumination), not the absolute number of rhodopsin molecules bleached...

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