Study of retinal dystrophy in RCS rats: a comparison of Mg-ATP dependent light scattering activity and ERG b-wave.

A comparative study using the techniques of ERG to measure the b-wave and light scattering relaxation spectrophotometry (LSRS) to determine the dynamic behavior Mg-ATP dependent processes in the rod photoreceptors of pigmented control and dystrophic RCS rats has been carried out. LSRS results, based exclusively on photoreceptor rod outer segment dynamics, suggest a progressive failure in the dark and light-induced Mg-ATP dependent processes as a function of age. The dark signal amplitude in the dystrophic rats decreases to about 50% of the control by 5 weeks post-natal; the light-induced signal has decreased by 30% in the same period. The ERG b-wave results indicate that the differences in the amplitude and the time required to attain the peak amplitude become increasingly pronounced between the control and dystrophic groups of rats again as a function of age. By 10 weeks of age, the intensity of light required to obtain a b-wave with a amplitude of 100 microM is 10(3) greater in the dystrophic RCS rats. Similarly, the time to achieve this peak increases in the dystrophs with age. These results indicate that the retinal dystrophy in the RCS rat affects the activity of the rod photoreceptor cells.

The activation of the cyclic-GMP phosphodiesterase via metarhodopsin I: a new model for vertebrate transduction.

Light-scattering spectrophotometry has been used to study rapid photo-induced molecular-cellular changes in vertebrate rod outer segments. Here, we discuss the temporal profiles of the nucleotide-independent P signal as a function of photobleaching, pH dependence in membrane-permeable and -impermeable buffers, angular and wavelength dependence, and cyclic-GMP phosphodiesterase inhibitors. On the basis of these observations, we suggest that (i) the P signal is coupled with the metarhodopsin I photo-intermediate and (ii) processes involved in the P signal invoke activation of cyclic-GMP phosphodiesterase. Furthermore, temperature-dependence studies indicate that the G protein does not participate in the scheme until the metarhodopsin II stage has been reached. This latter finding suggests that GTP-dependent processes are involved principally in the recovery of the system following light absorption. Our results point to a new model for phototransduction in vertebrate vision.

Light-induced interaction between rhodopsin and GTP-binding protein leads to the hydrolysis of GTP in the rod outer segment.

In the presence of exogenous GTP, vertebrate whole rod outer segments (ROS), with perforated plasma membranes in the "single particle" scattering range, elicit a light-induced light-scattering transient which we call the "G" signal. Here, we report on the characteristics of the "G" signal relative to the "binding" and "dissociation" signals reported by Kuhn and colleagues. Replacing GTP with guanylyl imidodiphosphate (GMP-PNP) does not give rise to the G signal. This indicates that hydrolysis of the terminal phosphate is required for the G signal and, in addition, GTP and GMP-PNP compete for the same binding site of the enzyme responsible for the G signal (i.e., GTP-binding protein). Also, neither GDP nor its nonhydrolyzable analogue, guanosine 5'-O-(2-thiodiphosphate), when present in ROS suspensions yield any light-scattering transient in the time period tested.

Cyclic GMP stimulation of a light-activated ATPase in rod outer segments.

Rod outer segments (ROSs) of vertebrate photoreceptor cells have been reported to contain several enzyme systems including a dark, Ca2+-stimulated ATPase, a rhodopsin kinase, a phosphodiesterase and a GTPase, all of which are light-stimulated. Recently, Thacher has found a light-stimulated Mg2+-ATPase in frog ROSs while our own laboratory has identified a dark, Ca2+-inhibited Mg2+-ATPase in bovine ROSs. Here we extend our observations on the Mg2+-ATPase and demonstrate that flash illumination following the dark ATPase process stimulated ATPase activity at a rate considerably faster than the dark process. In addition, we find that both the dark and light stimulated ATPase activities are markedly enhanced by cyclic GMP and inhibited by Ca2+.

A light scattering study on the ion permeabilities of dark-adapted bovine rod outer segment disk membranes.

The ion permeability properties of dark adapted bovine rod outer segment disk membranes were studied using light scattering to monitor osmotic responses of disks to various salts and ionophores. A preparation procedure is presented which provides very fresh rod outer segment material with mostly intact stacked disks, but with perforated plasma membrane. It is shown that in this preparation the disks (or rod sacs) are the only osmotically responding compartments and that these responses can be readily monitored by means of light-scattering techniques. The disk membrane is found under the conditions tested, to possess no measurable permeability to cations Na+, Ca2+, Mg2+ nor the the anions Cl-, Br-, NO3-, SO4(2-), H2PO4- and HPO4(2-). There is a considerable K+ permeability, which can be completely abolished by millimolar amounts of divalent cations. The proton permeability of the disk membrane is found to depend dramatically upon the preparation procedure and duration. The fresher the material used the lower is the proton permeability measured. In our freshest preparations, even after freeze-thawing in liquid nitrogen, the disks exhibit an H+ permeability which is so low that it cannot be measured with the techniques used in this study. Even in mitochondrial or chloroplast membmranes, in which proton gradients and therefore a low proton conductance play an essential role, such low proton permeabilities have not been found. This would suggest that proton gradients across the disk membrane could play an important role in the physiological function of the photoreceptor cell. In summary it can be said that the disk membrane, apparently more than any other natural membrane system studied so far, is capable of retaining ion gradients for extended periods of time.

Structural basis for the anticoagulant activity of heparin. 2. Relationship of anticoagulant activity to the thermodynamics and fluorescence fading kinetics of acridine orange-heparin complexes.

Complexing heparin or dermatan sulfate with the fluorescent probe acridine orange provides a means of studying electrostatic as well as static and dynamic conformational aspects of these glycosaminoglycans via the thermodynamic and photochemical (fluorescence fading) properties of these complexes. The cooperative binding constants (Kq), fluorescence fading rate parameters (r''), and anticoagulant activities of heparins fractionated according to anionic density all showed qualitatively the same dependence upon anionic density. When Kq and r'' were plotted against anticoagulant activity, empirical relationships were observed. Interestingly, the corresponding values for unfractionated dermatan sulfate fell on the lines defined by the heparin fractions. Temperature-dependence, studies demonstrated that differences in fading rate observed for heparins of different anionic densities are entropic in origin and reflect differences in the ability to assume a special configuration. Differences in activation entropy for fluorescence fading can be empirically correlated with anticoagulant activity. The latter correlation suggests a physical similarity in the roles played by anionic density in both fluorescence fading and anticoagulant activity.