Efficient single-photon detection by retinal rod photoreceptors requires timely and reproducible deactivation of rhodopsin. Like other G protein-coupled receptors, rhodopsin contains multiple sites for phosphorylation at its COOH-terminal domain. Transgenic and electrophysiological methods were used to functionally dissect the role of the multiple phosphorylation sites during deactivation of rhodopsin in intact mouse rods. Mutant rhodopsins bearing zero, one (S338), or two (S334/S338) phosphorylation sites generated single-photon responses with greatly prolonged, exponentially distributed durations. Responses from rods expressing mutant rhodopsins bearing more than two phosphorylation sites declined along smooth, reproducible time courses; the rate of recovery increased with increasing numbers of phosphorylation sites. We conclude that multiple phosphorylation of rhodopsin is necessary for rapid and reproducible deactivation.

The Ca2+-binding protein recoverin may regulate visual transduction in retinal rods and cones, but its functional role and mechanism of action remain controversial. We compared the photoresponses of rods from control mice and from mice in which the recoverin gene was knocked out. Our analysis indicates that Ca2+-recoverin prolongs the dark-adapted flash response and increases the rod's sensitivity to dim steady light. Knockout rods had faster Ca2+ dynamics, indicating that recoverin is a significant Ca2+ buffer in the outer segment, but incorporation of exogenous buffer did not restore wild-type behavior. We infer that Ca2+-recoverin potentiates light-triggered phosphodiesterase activity, probably by effectively prolonging the catalytic activity of photoexcited rhodopsin.

Changes in Young's modulus E (determined according to ASTM E-111) of polycrystalline pure iron deformed by a tensile test at room temperature are determined. From its original mean value, 210 GPa, E decreased with deformation to a mean value of 196 GPa at ϭ 0.060. Thereafter, slight recovery occurred and E stabilized to 198 GPa until the appearance of neck ( ϭ 0.100). With the aim to examine the causes of this behavior, residual stresses and textures were measured and the dislocation structure was observed by transmission electron microscopy (TEM). Longitudinal residual stresses increased from the first step of deformation ( ϭ 0.015) and remained constant until the samples fractured. There was no significant difference in texture throughout the deformation process, during which the increment of ␣ fiber was smooth. Thus, the decrease of E cannot be attributed to residual stresses or textures. A relationship between dislocation arrangement and decrease of E is proposed. Following the model established by Mott, dislocations can bow out in their glide planes, giving extra elastic strain and thus a decrease of E. The increase of the dislocation density during the first steps of deformation lowers the E values, since the extra elastic strain increases. At higher strains, when the cellular dislocation structure has formed (between ϭ 0.060 and 0.080), the dislocations in cell interiors are capable of giving an extra elastic strain, whereas the dislocations trapped in the cell walls are not. However, the dislocation density in cell interiors is lower than the dislocation density in the early stages of deformation in which the cell structure has not been developed. This produces the slight recovery of E measured at these strains. From ϭ 0.080, the values of E stabilized since no changes in dislocation density in cell interiors are observed.

Changes in the intracellular distribution of liver glycogen synthase (GS) might constitute a new regulatory mechanism for the activity of this enzyme at cellular level. Our previous studies indicated that incubation of isolated hepatocytes with glucose activated GS and resulted in its translocation from a homogeneous cytosolic distribution to the cell periphery. These studies also suggested a relationship with insoluble elements of the cytoskeleton, in particular actin. Here we show the translocation of GS in a different experimental model that allows the analysis of this phenomenon in long-term studies. We describe the reversibility of translocation of GS and its effect on glycogen distribution. Incubation of cultured rat hepatocytes with glucose activated GS and triggered its translocation to the hepatocyte periphery. The relative amount of the enzyme concentrated near the plasma membrane increased with time up to 8 h of incubation with glucose, when the glycogen stores reached their maximal value. The lithium-induced covalent activation of GS was not sufficient to cause its translocation to the cell periphery. The intracellular distribution of GS closely resembled that of glycogen. Our results showed an interaction between GS and an insoluble element of the hepatocyte matrix. Although no co-localization between actin filaments and GS was observed in any condition, disruption of actin cytoskeleton resulted in a significantly lower percentage of cells in which the enzyme translocated to the cell periphery in response to glucose. This observation suggests that the microfilament network has a role in the translocation of GS.

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Changes in the intracellular distribution of liver glycogen synthase (GS) might constitute a new regulatory mechanism for the activity of this enzyme at cellular level. Our previous studies indicated that incubation of isolated hepatocytes with glucose activated GS and resulted in its translocation from a homogeneous cytosolic distribution to the cell periphery. These studies also suggested a relationship with insoluble elements of the cytoskeleton, in particular actin. Here we show the translocation of GS in a different experimental model that allows the analysis of this phenomenon in long-term studies. We describe the reversibility of translocation of GS and its effect on glycogen distribution. Incubation of cultured rat hepatocytes with glucose activated GS and triggered its translocation to the hepatocyte periphery. The relative amount of the enzyme concentrated near the plasma membrane increased with time up to 8h of incubation with glucose, when the glycogen stores reached their maximal value. The lithium-induced covalent activation of GS was not sufficient to cause its translocation to the cell periphery. The intracellular distribution of GS closely resembled that of glycogen. Our results showed an interaction between GS and an insoluble element of the hepatocyte matrix. Although no co-localization between actin filaments and GS was observed in any condition, disruption of actin cytoskeleton resulted in a significantly lower percentage of cells in which the enzyme translocated to the cell periphery in response to glucose. This observation suggests that the microfilament network has a role in the translocation of GS.

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