Isolated Frog Rod Outer Segments Research Articles

Taurine neurons in rat hippocampal formation are relatively inert to cerebral ischemia.

It is well-known that transient cerebral ischemia causes selective loss of neurons in the hippocampus, striatum and cortical layers (14,24). It has been suggested that the difference in nerve cell vulnerability to ischemic damage is probably due to differences in the nature of their transmitters (14,22,27). For instances, GABAergic neurons in the striatum are more susceptible to transient ischemia than the cholinergic neurons (7) and dopaminergic neurons are more sensitive to ischemic damage than the GABAergic neurons in the gerbil brain (26). Since taurine has been demonstrated to have protective effects on the photoreceptor in the cat (12) and on the light-induced disruption of isolated frog rod outer segments (23), we decided to investigate the influence of transient cerebral ischemia on the taurine neurons in rat hippocampus. Taurine neurons were identified immunocytochemically with antibodies against cysteine sulfinic acid decarboxylase, the rate-limiting enzyme of taurine biosynthesis. Cysteine sulfinic acid decarboxylase has previously been used as a specific marker for identification of taurine neurons in the retina (10,17–19), cerebellum (3,4) and hippocampus (10,26,28).

Axial diffusion of retinol in isolated frog rod outer segments following substantial bleaches of visual pigment

Partial bleaches of rhodopsin were made in either the proximal or distal halves of isolated Rana pipiens rod outer segments. The fluorescence of all- trans retinol was recorded 15, 60 and 120 min following 17% and 63% bleaches. Some of the retinol that formed remained immobilized in the bleached halves of the outer segments, while another portion was slowly mobilized and diffused along the cell axis.

Taurine and hypotaurine inhibit light-induced lipid peroxidation and protect rod outer segment structure

Exposure of isolated frog rod outer segments to light (5000 lux) induces membrane disorganization and swelling. An increase of about 50% on lipid peroxidation, measured by the extent of malonaldehyde formation, accompanied the light-induced damage. Taurine and hypotaurine (25 mM) prevented the increase in lipid peroxidation, and provided an entire protection of rod outer segment structure.

Effect of light on Na-Ca exchange in rod outer segments in frog

The effect of illumination on the calcium (Ca) translocation mechanisms in isolated frog rod outer segments (ROS) was studied. The ATP-dependent Ca uptake and the Ca-Ca exchange mechanisms were unaffected by light. In contrast, we report a light-evoked Ca efflux which is mediated by the Na-Ca exchange system. The ratio of released Ca to rhodopsin bleaching was measured and the stoichiometry obtained was 5 Ca molecules released per mole of rhodopsin bleached. Concomitant to the Ca release, light induced Ca uptake, which increase the total Ca content of ROS. Physiological relevance of results to the phototransduction process is discussed.

Preparation and characterization of monoclonal antibodies to several frog rod outer segment proteins.

Monoclonal antibodies to proteins important in phototransduction in the frog rod outer segment have been obtained. These include 6 different antibodies to rhodopsin, 50 to a guanine nucleotide binding protein (G-protein; 40,000 daltons), and 2 to cytoplasmic proteins. The antigens used were Percoll-purified rod outer segments, a rod outer segment soluble protein fraction, or a soluble plus peripheral membrane protein fraction. Antibodies were assayed by solid phase assay using a fluorogenic detection system. Proteins to which antibodies bound were assayed on Western blots, and the sensitivities of three different detection systems were compared. Most antibodies bound to only one rod outer segment protein band on Western blots. Immunofluorescence microscopy demonstrated binding of both anti-rhodopsin and anti-G-protein to isolated frog rod outer segments. Antibodies were purified from either culture supernatants or ascites fluid on protein A affinity columns. Two purified anti-G-protein antibodies have binding affinities to 125I-labeled G-protein of less than 10(-6) M-1. Of 11 antibodies to frog or bovine G-protein tested in solid phase and Western blot assays, all bind to the alpha rather than the beta or gamma subunits. Procedures developed here are being used in preparing other antibodies that affect reactions in the phototransduction pathway.

Protective effect of taurine and zinc on peroxidation-induced damage in photoreceptor outer segments.

Exposure of isolated frog rod outer segments (ROS) to ferrous sulfate in a Krebs-bicarbonate medium causes a time-dependent disruption of the membrane organization of the discs. Ferrous sulfate also causes ROS swelling and aggregation. Addition of taurine (5-20 mM) and zinc sulfate (250 microM) to the incubation medium markedly protected ROS from the disrupting effect of ferrous sulfate. Of other amino acids tested, only beta-alanine had a protective effect on ROS structure. Ferrous sulfate caused an increase in lipid peroxidation, measured by malonaldehyde formation. The protective effect of taurine and zinc is not accompanied by a reduction of lipid peroxidation. Water accumulation occurs as a consequence of the peroxidative action of ferrous sulfate, and this effect was counteracted by taurine and zinc. Ferrous sulfate did not cause damage to ROS structure when incubation was carried out in sucrose-HEPES. Sodium, chloride, and bicarbonate ions caused ferrous sulfate to disrupt ROS structure. It is concluded that taurine and zinc protect ROS membranes from ion and/or water entry occurring as a consequence of membrane lipid peroxidation.

Taurine activation of a bicarbonate-dependent, ATP-supported calcium uptake in frog rod outer segments.

Isolated frog rod outer segments (ROS) with a leaky plasma membrane showed a bicarbonate-dependent, ATP-activated 45Ca accumulation. This calcium uptake requires magnesium and is specific for ATP; other nucleotides, ITP, GTP, UTP and the non-hydrolysable analogue of ATP beta-gamma-methylene ATP did not substitute for ATP. 45Ca accumulation was inhibited by mersalyl, ethylmaleimide, ruthenium red, oligomycin and dicyclohexylcarbodiimide and was unaffected by ouabain. Addiction of taurine to the incubation medium enhanced 45Ca uptake in a concentration-dependent manner; increases of more than 100% being produced by 25 mM taurine. The taurine-induced stimulation of 45Ca uptake was also sensitive to the tested inhibitors. The effect of taurine was only exerted on the bicarbonate-dependent, ATP-activated 45Ca uptake. Calcium accumulation observed in the absence of ATP or in a tris-buffered medium was unaffected by taurine. Other amino acids, glycine, GABA, beta-alanine, glutamic acid and the taurine analogue guanidinoethyl-sulfonate did not stimulate 45Ca uptake. These results suggest that taurine is affecting a Mg-ATPase activity responsible for calcium accumulation in frog ROS.

Protective effect of taurine on the light‐induced disruption of isolated frog rod outer segments

Isolated frog rod outer segments (ROS) incubated in a Krebs-bicarbonate medium, and illuminated for 2 h, show a profound alteration in their structure. This is characterized by distention of discs, vesiculation, and a marked swelling. The light-induced ROS disruption requires the presence of bicarbonate and sodium chloride. Replacement of bicarbonate by TRIS or HEPES protects ROS structure. Also, substitution of sodium chloride by sucrose or choline chloride maintains unaltered the ROS structure. Deletion of calcium, magnesium, or phosphate does not modify the effect produced by illumination. An increased accumulation of labeled bicarbonate and tritiated water is observed in illuminated ROS, as compared with controls in the dark. The presence of taurine, GABA, or glycine, at concentrations of 5-25 mM, effectively counteracts the light-induced ROS disruption. Taurine (25 mM) reduces labeled bicarbonate and tritiated water levels to those observed in the dark incubated ROS.

On the evaluation of photoreceptor properties by micro-fluorimetric measurements of fluorochrome diffusion.

By use of the microfluorimetric technique it is possible to study the diffusion of the fluorochrome di-dansylcystine (DDC) within isolated frog rod outer segments (ros) which are immobilysed in agarose gel. For this purpose, by a short hypotonic shock a leak is applied to one end of the ros. By this open end the DDC enters the rod and migrates through the whole outer segment. Following the propagation of the fluorescence boundary with time the cytoplasmatic diffusion constant can be determined if a chromatographic model is used to allow for the considerable binding of DDC to the inner membrane surface. With a binding constant K = 5 . 10(-4) cm the cytoplasmatic diffusion constant was found to be D = 1.3 . 10(-6) cm2/s whereas Dg = 2 . 10(-6 cm2/s and Dr = 3.5 . 10(-6) cm2/s were found in agarose gel or ringer solution, respectively. Using the mobility reduction factor given by D/Dr approximately equal to 0.4 to calculate the cytoplasmatic conductivity an inner resistance per length of 1.7 M omega/mu could be calculated for a frog rod which is in good agreement with corresponding data obtained from electrophysiological measurements.

Influence of light and calcium on guanosine 5'-triphosphate in isolated frog rod outer segments.

Frog rod outer segments contain approximately 0.25 mol of GTP and 0.25 mol of ATP per mol of rhodopsin 3 min after their isolation from the retina. UTP and CTP are present at 10-fold and 100-fold lower levels, respectively. Concentrations of GTP and ATP decline in parallel over the next 4 min to reach relatively stable levels of 0.1 mol per mol of rhodopsin. Illumination reduces the concentration of endogenous GTP but not ATP. This light-induced decrease in GTP can be as large as 70% and has a half-time of 7 s. GTP is reduced to steady intermediate levels during extended illumination of intermediate intensity, but partially returns to its dark-adapted level after brief illumination. The magnitude of the decrease increases as a linear function of the logarithm of continuous light intensity at levels which bleach between 5 X 10(2) and 5 X 10(6) rhodopsin molecules/outer segment per second. This exceeds the range of intensities over which illumination causes decreases in the cyclic GMP content and permeability of isolated outer segments (Woodruff and Bownds. 1979. J. Gen. Physiol. 73:629-653). Thus, over 4 log units of light intensity, a sensitivity control mechanism functions to make extended illumination less effective in stimulating a GTP decrease. GTP levels in dark-adapted outer segments are sensitive to changes in calcium concentration in the suspending medium. If the external calcium concentration is reduced to 10(-8) M, GTP concentration is lowered to the same level caused by saturating illumination, and the GTP remaining is no longer light-sensitive. Lowering calcium concentration to intermediate levels between 10(-6) and 10(-8) M reduces GTP to stable intermediate levels, and the GTP remaining can be reduced by light. Restoration of millimolar calcium drives synthesis of GTP, but not of ATP, and GTP lability towards illumination is again observed. These calcium-induced changes in GTP are diminished by the addition of the divalent cation ionophore A23187. Lowering or raising magnesium levels does not influence the GTP concentration. These data raise the possibility that light activates either a calcium transport mechanism driven by the hydrolysis of GTP, or some other calcium-sensitive GTPase activity of unknown function. Known light-dependent reactions involving cyclic nucleotide transformations and rhodopsin phosphorylation appear to account for only a small fraction of the light-induced GTP decrease.

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

The isolation of stable cattle rod outer segments with an intact plasma membrane

A procedure is described to purify and stabilize cattle rod outer segments with an intact plasma membrane. Three criteria are applied to assess the integrity of the latter. Upon photolysis in these rod outer segments: (1) exogenous ATP cannot phosphorylate rhodopsin located in the disk membrane. (2) Endogenous cofactors (NADPH, NADPH-regenerating system) are still available in the rod cytosol and consequently retinol is the final photoproduct of photolysis of rhodopsin. (3) The rod cytosol can maintain a pH different from that of the medium, since the later stages of rhodopsin photolysis are independent of the medium pH. The stability and homogeneity of the preparation appear to be much better than those of freshly isolated frog rod outer segments, which have been used most frequently so far for experiments on the physiology of rod outer segments. In addition, these cattle rod outer segments remain intact during various manipulations and therefore considerably extend the experimental possibilities when intact rod outer segments are required.

Photoresponses of isolated frog rod outer segments

Photoresponses were recorded from single isolated frog rod outer segments with and without myoid by sucking them into the tip of a tight-fitting glass pipet which served as a recording electrode. The size and behavior of the photovoltage indicate that it is probably due to a proximally flowing external photocurrent causing a potential drop across the medium between the cell and the pipet wall. Further properties of the response, including the response amplitude-stimulus intensity curve and some adaptation results are discussed.

Local stimulation and local adaptation of single isolated frog rod outer segments

Local stimulation and local adaptation of single isolated frog rod outer segments

Local stimulation and local adaptation of single isolated frog rod outer segments

Local stimulation and local adaptation of single isolated frog rod outer segments

Immunocytochemical localization of a large intrinsic membrane protein to the incisures and margins of frog rod outer segment disks.

Immunocytochemical techniques have localized a large protein which is an intrinsic membrane component of isolated frog rod outer segments (ROS). This large protein whose apparent mol wt is 290,000 daltons comprises about 1--3% of the ROS membrane mass. Its molar ratio to opsin is between 1:300 and 1:900. Adequate immune responses were obtained with less than 30 microgram (100 pmol) of antigen per rabbit. Antibodies to the large protein were used for its localization on thin sections of frog retina embedded in glutaraldehyde cross-linked bovine serum albumin (BSA). Specifically bound antibodies were detected by an indirect sequence with ferritin-conjugated antibodies. This technique detected the protein which is represented by 1,000--3,000 molecules per disk. This indicates that the procedure is sufficiently sensitive for analysis of membrane components in low molar proportions. The large protein was specifically localized to the incisures of ROS disks which divide the disks into lobes and to the disk margin. Thus, opsin is mobile within the membrane of the disk while the large protein is apparently constrained to the disk edges. This finding raises the possibility that special functions are also localized ot his unusual region of high curvature, and that collisions of bleached opsin with these edges are physiologically important in couter segment function.

Light-sensitive swelling of isolated frog rod outer segments as an in vitro assay for visual transduction and dark adaptation.

Frog rod outer segments swell slowly after being shaken from an excised retina into a modified Ringer's solution. The swelling has the following characteristics: (a) It is suppressed by illumination which bleaches only 500 rhodopsin molecules per outer segment per second. This is approximately the level required to saturate the in vivo receptor potential. (b) Light suppression is seen in NaCl but not in KCl solutions. (c) Dark swelling is labile and is enhanced by calf serum, low calcium concentrations, dithiothreitol, and cyclic nucleotide phosphodiesterase inhibitors. (d) Lowering the pH to 5.5 or removing magnesium reversibly reduces dark swelling to the same extent as illumination. (e) The amount of light required for maximal suppression of dark-swelling increases approximately 10-fold if the calcium concentrations is lowered by EGTA addition. (f) The effect of illumination is irreversibly abolished by antimycin and other inhibitors of mitochondrial electron transport. (g) A process analogous to dark adaptation in vivo can be observed: If 10-50% of the rhodopsin present is bleached and the outer segments are then kept dark, rapid dark swelling returns after a period of 15-45 min. This swelling is again sensitive to light. We tentatively ascribe the light suppression of swelling to the same decrease in sodium permeability which is observed on illuminating living receptor cells. The experiments suggest that outer segments retain their competence to perform both transduction and dark adaptation after their separation from the retina.

Biochemical aspects of the visual process. XXV. Light-induced calcium movements in isolated frog rod outer segments

Suspensions of isolated rod outer segments are shown to have a high calcium content of up to 11 moles calcium per mole rhodopsin. Osmotic lysis of the outer segments demonstrates the presence of two calcium fractions, a soluble one and a particulate one. The particulate fraction apparently coincides with the rod disks or with disk fragments. Illumination of intact rod outer segments in calcium-free ATP-containing Ringer solution has no measurable effect upon their total caclium content, but causes a significant shift of calcium from the particulate to the soluble fraction. This light effect is retained in lysed outer segments resuspended in calcium-free ATP-containing Ringer. These results support a function of calcium as a transmitter in light transduction in rod outer segments.

Changes in structure of the disks of retinal rods in hypotonic solutions.

ABSTRACT Changes in isolated frog rod outer segments, suspended in hypotonic solutions, have been examined by light and electron microscopy. Swelling of the disk occurs in hypotonic solutions. When one half or more NaCl is omitted from the Ringer solution used for suspending the rod outer segments, swelling is accompanied by the appearance of localized, irregular expansions projecting as buds from the disks. The axes of the buds tend to be in the plane of the disk, as can be seen in cross-sections of outer segments. In longitudinal sections of outer segments, the sectioned buds have profiles which were previously interpreted as vesicles. Attention is drawn to the properties of the disk edge, among which is a resistance to extension.

Membrane characteristics and osmotic behavior of isolated rod outer segments.

Freshly isolated frog rod outer segments are sensitive osmometers which retain their photosensitivity; their osmotic behavior reveals essentially the same light-sensitive Na(+) influx observed electrophysiologically in the intact receptor cell. Using appropriate osmotic conditions we have examined freeze-etch replicas of freshly isolated outer segments to identify the membrane which regulates the flow of water and ions. Under isosmotic conditions we find that the disc to disc repeat distance is almost exactly twice the thickness of a disc. This ratio appears to be the same in a variety of vertebrate rod outer segments and can be reliably measured in freeze-etch images. Under all our osmotic conditions the discs appear nearly collapsed. However, when the length of the outer segment is reduced by hyperosmotic shocks the discs move closer together. This markedly reduces the ratio of repeat distance to disc thickness since disc thickness remains essentially constant. Thus, the length reduction of isolated outer segments after hyperosmotic shocks primarily results from reduction of the extradisc volume. Since the discs are free floating and since they undergo negligibly small changes in volume, the plasma membrane alone must be primarily responsible for regulating the water flux and the light-sensitive Na(+) influx in freshly isolated outer segments. On this basis we calculate, from the osmotic behavior, that the plasma membrane of frog rod outer segment has a Na(+) permeability constant of about 2.8 x 10(-6) cm/s and an osmotic permeability coefficient of greater than 2 x 10(-3) cm/s.