Molecular characterization of a third member of the guanylyl cyclase-activating protein subfamily.
(1/2708)
The mammalian retina contains at least two guanylyl cyclases (GC1 and GC2) and two guanylyl cyclase-activating proteins (GCAP1 and GCAP2). Here we present evidence of the presence of a new photoreceptor-specific GCAP, termed GCAP3, which is closely related to GCAP1. The sequence similarity of GCAP3 with GCAP1 and GCAP2 is 57 and 49%, respectively. Recombinant GCAP3 and GCAP2 stimulate GC1 and GC2 in low [Ca2+]free and inhibit GCs when [Ca2+]free is elevated, unlike GCAP1, which only stimulates GC1. GCAP3 is encoded by a distinct gene present in other mammalian species but could not be detected by genomic Southern blotting in rodents, amphibians, and lower vertebrates. The intron/exon arrangement of the GCAP3 gene is identical to that of the other GCAP genes. While the GCAP1 and GCAP2 genes are arranged in a tail-to-tail array on chromosome 6p in human, the GCAP3 gene is located on 3q13.1, suggesting an ancestral gene duplication/translocation event. The identification of multiple Ca2+-binding proteins that interact with GC is suggestive of complex regulatory mechanisms for photoreceptor GC. (+info)
Gap junctions in the differentiated neural retinae of newly hatched chickens.
(2/2708)
Gap junctions in the neural retinae of newly hatched chickens were examined in thin section and by freeze cleaving. Unusual gap junctions containing linear arrays of intramembrane particles are found between principal and accessory cones which form a double cone at the region of the outer limiting membrane. These unusual gap junctions are often continuous with macular aggregates of hexagonally packed intramembrane particles which are characteristic of a typical gap junction. Typical gap junctions are also found in both the outer and the inner plexiform layers and in the outer nuclear layer, but are not so abundant as in the outer limiting membrane region. The sizes of intramembrane particles and their centre-to-centre spacing within the macular aggregate of a gap junction in differentiated neural retinae are slightly larger than those in undifferentiated neural retinae. Tight junctions are not found in differentiated neural retinae. (+info)
Hypersensitivity in the anterior median eye of a jumping spider.
(3/2708)
Changes in sensitivity of the photoreceptor cells of the anterior median eye of the jumping spider Menemerus confusus Boes. et Str. have been studied by recording electroretinograms (ERGs) and receptor potentials. The amplitudes of the responses (ERGs and receptor potentials) increase during repetitive stimulation, with a maximum increase at 3-5 s intervals. The sensitivity of the photoreceptor cell is greater for about 60 s following illumination (maximum magnitude at 3-5 s) than it is during complete dark adaptation. This phenomenon, which we call 'hypersensitivity', is lost within one day following surgery in physiological saline. Upon loss of hypersensitivity, the sensitivity decrease during light adaptation is greater than for the normal eye and the small increase of sensitivity following the onset of illumination observed for the normal eye is lost. (+info)
The relation between intercellular coupling and electrical noise in turtle photoreceptors.
(4/2708)
1. Intracellular recordings from cones and rods in the retina of the turtle, Pseudemys scripta elegans, revealed that in darkness the cell voltage fluctuated spontaneously about its mean level. The fluctuations were reduced during bright steady illmination of the cell often to a level close to that obtained with the electrode outside the cell where the noise did not change significantly during illumination. 2. The magnitude of the intrinsic dark noise (voltage variance in darkness minus voltage variance in strong light) varied widely from cell to cell. In the noisiest cones it was about 0-4 mV2 while in quiet cones it was often as low as 0-01 mV2. The noise appeared radom and could be fitted by a Gaussian probability density function. 3. The spread of voltage in the network of coupled photoreceptors was estimated by measuring the spatial profile of the response to a brief flash of constant intensity moved across the retina. For a light stimulus in the form of a long narrow slit, the peak flash response usually decayed exponentially with displacement from the centred position. 4. For maximum responses less than about 5 mV in cones, the length constant of exponential decay, lambda, varied from less than 10 mum to greater than 35 mum, and the values obtained in opposite directions were often unequal. Background illumination did not significantly change lambda. In cells with extremely narrow spatial profiles, an exponential fit to the decay could not be made reliably. 5. Occasionally the spatial profiles had definite secondary peaks. In the most pronounced examples in a red-sensitive cone and in a rod the maxima were separated by about 20 and 50 mum respectively; for each, one peak was approximately as sharp as the optical stimulator while the second was broader. 6. Cones with short length constants displayed high dark noise while cones with long length constants were relatively quiet. 7. Three models of electrical coupling between cells were investigated: one based on a distributed network, one on a discrete square grid arrangement, and one on a discrete hexagonal array. Each model predicts a strong dependence of both noise and input resistance on length constant, and for tightly coupled cells each predicts that voltage variance is proportional to lambda-2. 8. The measured relationship between voltage variance and lambda in a large sample of cones was well described by both discrete models when the average cell spacing was taken to be approximately 15 mum. 9... (+info)
On the analysis of nerve signals deduced from metacontrast experiments with human observers.
(5/2708)
1. This paper reviews Alpern, Rushton & Torii's (1970a-d) derivation of the size of the inhibitory nerve signal arising from after flashes in the metacontrast experiment. 2. Their geometric argument is recast in terms of simple functional equations. This form of argument clearly displays the role of their assumptions in obtaining their main conclusion: nerve signal is linear in intensity over a range of 3-4 log units. 3. Two disadvantages of their approach are discussed. First, it is noted that in the presence of the data the assumption they employ in their analysis is logically equivalent to their conclusion. 4. Secondly, accepting their claim that the nerve signal generated by the after flash is linear over a broad range of intensities, and that this inhibitory signal simply cancels the excitatory signal of the test flash, leads to the conslusion that over this same intensity range the excitatory nerve signal is a power function with an exponent of close to two. This is incompatible with the suggestion that photoreceptor signals have been measured. (+info)
Iron-induced cytotoxicity in cultured rat retinal neurons.
(6/2708)
Oxidative stress has been proposed as a major injury mechanism in the central nervous system including the retina. In this study, as an initial attempt to study the mechanism of oxidative injury in the retina, we developed a cell culture model by utilizing the iron exposure paradigm. Exposure of rat retinal cultures for 24 hours to 10-40 MicroM ferrous or ferric chloride induced a concentration-dependent death of retinal neurons but not of photoreceptors or astrocytes. An antioxidant, trolox effectively attenuated the iron-induced death of neurons and photoreceptors in a dose-dependent manner whereas neither glutamate receptor antagonists nor cycloheximide were protective. Of retinal interneurons, GABAergic neurons were more vulnerable to the iron toxicity than calbindin (+) horizontal neurons. These findings show that iron exposure induces anti-oxidant-sensitive neuronal injury in retinal culture, independent of the excitotoxic or the apoptotic mechanisms. Of retinal neurons, different cell types exhibit differential vulnerabilities to the iron-induced oxidative injury. This simplified culture model system may be useful in elucidating mechanisms of oxidative injury in the retina. (+info)
Mapping functional domains of the guanylate cyclase regulator protein, GCAP-2.
(7/2708)
Guanylate cyclase regulator protein (GCAP)-2 is a Ca2+-binding protein that regulates photoreceptor outer segment membrane guanylate cyclase (RetGC) in a Ca2+-sensitive manner. GCAP-2 activates RetGC at free Ca2+ concentrations below 100 nM, characteristic of light-adapted photoreceptors, and inhibits RetGC when free Ca2+ concentrations are above the 500 nM level, characteristic of dark-adapted photoreceptors. We have mapped functional domains in GCAP-2 by using deletion mutants and chimeric proteins in which parts of GCAP-2 were substituted with corresponding fragments of other closely related recoverin-like proteins that do not regulate RetGC. We find that in addition to the EF-hand Ca2+-binding centers there are three regions that contain GCAP-2-specific sequences essential for regulation of RetGC. 1) The region between Phe78 and Asp113 determines whether GCAP-2 activates outer segment RetGC in low or high Ca2+ concentrations. Substitution of this domain with the corresponding region from neurocalcin causes a paradoxical behavior of the chimeric proteins. They activate RetGC only at high and not at low Ca2+ concentrations. 2) The amino acid sequence of GCAP-2 between Lys29 and Phe48 that includes the EF-hand-related motif EF-1 is essential both for activation of RetGC at low Ca2+ and inhibition at high Ca2+ concentrations. Most of the remaining N-terminal region can be substituted with recoverin or neurocalcin sequences without loss of GCAP-2 function. 3) Region Val171-Asn189, adjacent to the C-terminal EF-4 contributes to activation of RetGC, but it is not essential for the ability of Ca2+-loaded GCAP-2 to inhibit RetGC. Other regions of the molecule can be substituted with the corresponding fragments from neurocalcin or recoverin, or even partially deleted without preventing GCAP-2 from regulating RetGC. Substitution of these three domains in GCAP-2 with corresponding neurocalcin sequences also affects activation of individual recombinant RetGC-1 and RetGC-2 expressed in HEK293 cells. (+info)
Mapping sites in guanylyl cyclase activating protein-1 required for regulation of photoreceptor membrane guanylyl cyclases.
(8/2708)
Guanylyl cyclase activating protein (GCAP)-1 regulates photoreceptor membrane guanylyl cyclase, RetGC, in a Ca2+-sensitive manner. It contains four Ca2+-binding motifs, EF-hands, three of which are capable of binding Ca2+. GCAP-1 activates RetGC in low Ca2+ and inhibits it in high Ca2+. In this study we used deletion and substitution analysis to identify regions of GCAP-1 sequence that are specifically required for inhibition and activation. A COOH-terminal sequence within Met157 to Arg182 is required for activation but not for inhibition of RetGC. We localized one essential stretch to 5 residues from Arg178 to Arg182. Another sequence essential for activation is within the N-terminal residues Trp21 to Thr27. The region between EF-hands 1 and 3 of GCAP-1 also contains elements needed for activation of RetGC. Finally, we found that inhibition of RetGC requires the first 9 amino-terminal residues of GCAP-1, but none of the residues from Gln33 to the COOH-terminal Gly205 are specifically required for inhibition. The ability of GCAP-1 mutants to regulate RetGC was tested on total guanylyl cyclase activity present in rod outer segments. In addition, the key mutants were also shown to produce similar effects on recombinant bovine outer segment cyclases GC1 and GC2. (+info)