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Multiple phosphorylation of rhodopsin and the in vivo chemistry underlying rod photoreceptor dark adaptation.

Dark adaptation requires timely deactivation of phototransduction and efficient regeneration of visual pigment. No previous study has directly compared the kinetics of dark adaptation with rates of the various chemical reactions that influence it. To accomplish this, we developed a novel rapid-quench/mass spectrometry-based method to establish the initial kinetics and site specificity of light-stimulated rhodopsin phosphorylation in mouse retinas. We also measured phosphorylation and dephosphorylation, regeneration of rhodopsin, and reduction of all-trans retinal all under identical in vivo conditions. Dark adaptation was monitored by electroretinography. We found that rhodopsin is multiply phosphorylated and then dephosphorylated in an ordered fashion following exposure to light. Initially during dark adaptation, transduction activity wanes as multiple phosphates accumulate. Thereafter, full recovery of photosensitivity coincides with regeneration and dephosphorylation of rhodopsin.

Adaptation, Ocular↗

A comparison of the second harmonic generation from light-adapted, dark-adapted, blue, and acid purple membrane.

The second order nonlinear polarizability and dipole moment changes upon light excitation of light-adapted bacteriorhodopsin (BR), dark-adapted BR, blue membrane, and acid purple membrane have been measured by second harmonic generation. Our results indicate that the dipole moment changes of the retinal chromophore, delta mu, are very sensitive to both the chromophore structure and protein/chromophore interactions. Delta mu of light-adapted BR is larger than that of dark-adapted BR. The acid-induced formation of the blue membrane results in an increase in the delta mu value, and formation of acid purple membrane, resulting from further reduction of pH to 0, returns the delta mu to that of light-adapted BR. The implications of these findings are discussed.

Adaptation, Physiological↗

Tryptamine and some related molecules block the accumulation of a light-sensitive pool of cyclic AMP in the dark-adapted, dark-incubated mouse retina.

Dark-adapted retinas of mice (C57BL/6J) incubated in the dark in media containing 1 mM 3-isobutylmethylxanthine (IBMX) or 5 mM Co2+ accumulate cyclic AMP (cAMP). A portion of this pool is light sensitive, as light can prevent or reverse its accumulation. Similarly, tryptamine, serotonin, 5-methoxytryptamine, bufotenine, and 5-methoxydimethyltryptamine can block the accumulation of the light-sensitive pool of cAMP, whereas tryptophan, melatonin, N-acetylserotonin, 5-methoxytryptophol, and tetrahydro-beta-carbolines are inactive. The phenomenon is not seen with mutant mouse retinas (rd/rd), which lack most photoreceptors, but persists in abnormal retinas containing photoreceptors but with extensive neuronal depletion in the inner retina. Tryptamine also inhibits cAMP accumulation in either dark or light-adapted retinas exposed to forskolin alone but not in media containing high levels of forskolin plus 1 mM IBMX. There is some suggestion that serotonin 5-HT-2 antagonists can partially reverse the action of the tryptamines, but hitherto undescribed receptors may be involved. Current data suggest that photoreceptors are the target for the action of the tryptamines.

1-Methyl-3-isobutylxanthine↗

Light adaptation and dark adaptation of human rod photoreceptors measured from the a-wave of the electroretinogram.

1. We recorded the a-wave of the human electroretinogram from subjects with normal vision, using a corneal electrode and ganzfeld (full-field) light stimulation. From analysis of the rising phase of rod-isolated flash responses we determined the maximum size (amax) of the a-wave, a measure of the massed circulating current of the rods, and the amplification constant (A) of transduction within the rod photoreceptors. 2. During light adaptation by steady backgrounds the maximal response was reduced, as reported previously. amax declined approximately as I0/(I0 + IB), where IB is retinal illuminance and I0 is a constant. In different subjects I0 ranged from 40 to 100 trolands, with a mean of 70 trolands, corresponding to about 600 photoisomerizations s-1 per rod. (1 troland is the retinal illuminance that results when a surface luminance of 1 cd m-2 is viewed through a pupil area of 1 mm2.) The amplification constant A decreased only slightly in the presence of steady backgrounds. 3. Following a full bleach amax recovered along an S-shaped curve over a period of 30 min. There was no detectable response for the first 5 min, and half-maximal recovery took 13-17 min. 4. The apparent amplification constant decreased at early times after large bleaches. However, upon correction for reduced light absorption due to loss of pigment, with regeneration of rhodopsin occurring with a time constant of 9-15 min in different subjects, it appeared that the true value of A was probably unchanged by bleaching. 5. The recovery of amax following a bleach could be converted into recovery of equivalent background intensity, using a 'Crawford transformation' derived from the light adaptation results. Following bleaches ranging from 10 to > 99 %, the equivalent background intensity decayed approximately exponentially, with a time constant of about 3 min. 6. The time taken for amax to recover to a fixed proportion of its original level increased approximately linearly (rather than logarithmically) with fractional bleach, with a slope of about 12 min per 100 % bleach. Similar behaviour has previously been seen in psychophysical dark adaptation experiments, for the dependence of the 'second component' of recovery on the level of bleaching.

Adaptation, Ocular↗

Relation of brightness to threshold for light-adapted and dark-adapted rods and cones: effects of retinal eccentricity and target size.

'Equal-brightness' functions of retinal eccentricity and target diameter were measured by a matching procedure, and compared with the corresponding threshold functions for four different adaptation conditions: light-adapted cones (LAC), dark-adapted cones (DAC), light-adapted rods (LAR) and dark-adapted rods (DAR). The separation between log brightness matches and log thresholds decreased with eccentricity and increased with target size for all adaptation conditions, but overall separation was substantially greater for the DAR condition than for the other three. A two-channel model of achromatic brightness is proposed to explain the results. The model assumes 'strong' and 'weak' channels, which contribute unequally to brightness. These channels are tentatively identified with tonic and phasic classes of retinal ganglion cells.

Adaptation, Ocular↗

Dark adaptation in diabetics.

Dark adaptation in diabetics was recorded with Goldmann-Weeker's adaptometer and analysed according to the stages of retinopathy by Scott. Dark adaptation curves in diabetics were divided into three groups; normal dark adaptation; subnormal one which showed normal first curve of dark adaptation and impaired second curve; abnormal dark adaptation which showed impaired dark adaptation in the whole course. The cases with normal dark adaptation were found to be few in the stages Ia, II and IIIa of retinopathy. Subnormal dark adaptation was rarely found in the stages Ia, II, IIIa and IIb of retinopathy. Abnormal dark adaptation was recorded in most patients with diabetic retinopathy and found in all th stages of retinopathy. A 5% level of significance was statistically recognized in the thresh old at 35 min after light adaptation between the stages IIIa and IIIb of retinopathy. From this viewpoint, significance in severity of diabetic retinopathy was discussed.

Dark Adaptation↗

The separation of cone mechanisms in dark adaptation.

1. In dark adaptation the threshold is raised as though the bleaching of visual pigment generated an equivalent background. Now Stiles has shown that real coloured backgrounds act selectively upon the various colour mechanisms, so we ask: ;Do equivalent backgrounds from coloured bleachings also act selectively?'2. When dark adaptation was plotted using a blue test flash (Fig. 1b) following bleaching by orange light a kinked curve was obtained. The upper branch was shown to have the same dark adapted threshold as Stiles blue (pi(1)) mechanism and the lower branch as his green (pi(4)) mechanism. The pi(4) dark adaptation curve alone (unkinked) was obtained using a white instead of an orange bleach.3. Dark adaptation curves were obtained in which the test flash was presented upon various steady backgrounds. In conditions where only pi(4) was involved (Fig. 3) the experimental results fitted the curves calculated on the assumption that the equivalent background of bleaching simply adds to the real background in raising the threshold-a condition already established for rods.4. In conditions where pi(4) and pi(1) were both present (blue test, yellow-green background and white bleach) kinked dark adaptation curves were obtained (Fig. 2) where the upper branch (pi(4)) coincided with those of Fig. 3 and the lower were due to pi(1).5. The blue mechanism recovers in dark adaptation at about the same rate as red and green, or slightly slower.6. Dark adaptation curves with red (pi(5)) and green (pi(4)) limbs can be obtained after a deep red bleach (Fig. 4) using a red test flash and a green background. The red and the green limbs were also plotted alone in their entirety by slightly changing the conditions.7. We are led to the idea of three colour mechanisms that adapt as independently one of another after bleaching as they do with backgrounds.8. Though this simple independence accounts for the wide and conspicuous range of adaptive phenomena, we have encountered some special conditions (not here described) that seem to imply a measure of interaction between the different colour mechanisms.

Adaptation, Ocular↗

Intracellular recordings of rod responses during dark-adaptation.

1. Dark-adaptation of rod photoreceptors has been studied in the isolated axolotl (Ambystoma mexicanum) retina by intracellular recordings. Rod responsiveness was greatly reduced immediately after a 30 sec partial bleach, but partially recovered with time in the dark. 2. In parallel spectrophotometric measurements using isolated retinas, regeneration of the rod pigment could not be detected after a 30 sec bleach. 3. During rod dark-adaptation, the response of a rod to a given stimulus increased in amplitude, duration, and rate of rise but did not recover completely to the dark-adapted values. Response latency was lengthened immediately after a bleach but ultimately returned to the dark-adapted level. 4. The time courses of dark-adaptation determined on the basis of the intensity of a stimulus needed to evoke a response having a criterion amplitude, a criterion duration, or a criterion rate of rise were similar. On the other hand changes in latency of the response and magnitude of the saturated amplitude followed different time courses. Change in log threshold was found to be related to change in saturated amplitude by an exponential function during dark-adaptation. 5. After bleaching 10% or less of the rod pigment, the kinetics of both recovery of log threshold and decrease in absorbance at 400 nm (metarhodopsin II+free retinal) could be described by two concurrent first-order processes having similar time constants. However, after bleaching more than 10% of the rod pigment, changes in sensitivity and absorbance did not follow parallel time courses. 6. Metarhodopsin III cannot be solely responsible for setting the axolotl rod sensitivity since rod thresholds decrease monotonically during dark-adaptation whereas meta III concentration reaches a peak 3 min after the bleach and decreases thereafter.

Ambystoma↗

On the photocycle and light adaptation of dark-adapted bacteriorhodopsin.

Pulsed Nd laser (25 ns, 530 nm) photolysis experiments were carried out at room temperature in aqueous suspensions of dark- and light-adapted fragments of the purple membrane of Halobacterium halobium. It is shown that the (50%) 13-cis isomeric component (BR13-cis) of dark-adapted bacteriorhodopsin (BRDA) undergoes a photocycle involving a characteristic transient absorbing in the neighborhood of 610 nm. At relatively high excitation intensities BR13-cis is converted to the same 410 nm (M) transient that characterized the photocycle of the all-trans isomer (BRtrans) of light-adapted bacteriorhodopsin (BRLA). This process, which competes with the generation of the "610" species, is attributed to the photo-induced conversion, during the pulse, of BR13-cis (or of its primary photoproduct "X") to a species in the BRtrans photocyte. The relationship between these observations and the mechanism of BRDA hv leads to BRLA adaptation at low excitation intensities (for which a quantum yield limit, 0 less than or equal to (3.5 +/- 0.7) X 10(-2) , is established) is discussed.

Bacteriorhodopsins↗

Photoconversion from the light-adapted to the dark-adapted state of bacteriorhodopsin.

Dark and light adaptation of bacteriorhodopsin in purple membrane multilayers at less than 100% relative humidity differs from that seen in suspensions. Equilibrium between the two bacteriorhodopsin isomers (bR cis 550 and bR trans 570) in the light-adapted state becomes dependent on the wavelength of actinic light. Excitation at the red edge of the visible absorption band causes dark adaptation in a light-adapted sample. Using polarized actinic and measuring light, we show that acceleration of the dark adaptation through heating by actinic light cannot explain this observation. A light-driven bR trans 570 to bR cis 550 reaction that competes with the well-known 13 cis-to-all-trans light adaptation reaction must exist under our experimental conditions. Trans-to-cis conversion is a one-photon process distinct from the two photon process observed by others in purple membrane suspensions (Sperling, W., C. N. Rafferty, K. D. Kohl, and N. A. Dencher, 1978, FEBS (Fed. Eur. Biochem. Soc.) Lett. 97:129-132). Its quantum efficiency increases monotonously on reducing the hydration level, and is paralleled by an increase in the lifetime of the M410 intermediate of the trans photocycle. We suggest that at this point a branch leads from the all-trans into the 13-cis photocycle. It is probably the same reaction that causes the reduced light adaptation in monomeric bacteriorhodopsin (Casadio, R., H. Gutowitz, P. Mowery, M. Taylor, and W. Stoeckenius, 1980, Biochim. Biophys. Acta. 590:13-23; Casadio, R., and W. Stoeckenius, 1980, Biochemistry. 19:3374-3381).

Bacteriorhodopsins↗

Evidence for two sites of adaptation affecting the dark-adapted ERG of cats and primates.

The present study compared the effects of full-field steady adapting backgrounds on the sensitivity of the scotopic threshold response (STR) of the dark-adapted ERG and scotopic PII (b-wave and d.c.-component) to Ganzfeld flashes in cats (n = 4), macaque monkeys (n = 2), and one human subject. In cats, the sensitivity of the STR was reduced by a factor of 2 by backgrounds that were 500 times weaker than backgrounds reducing PII; and for the primates, the STR was reduced by backgrounds almost 100 times weaker than those reducing PII. Since the STR is generated more proximally in the retina than PII, these results provide evidence for proximal and more distal retinal sites of postreceptoral light adaptation. A practical implication is that dim scattered room light can remove the STR from the ERG while hardly affecting PII.

Adaptation, Ocular↗