Intact Rod Research Articles

Retinol Kinetics in the Isolated Retina Determined by Retinoid Extraction and HPLC

Suzuki et al. [Vis. Res.26, 425–9 (1986);Vis. Res.28, 1061–70 (1988)] have described a formaldehyde-based (HCHO-based) extraction procedure that efficiently recovers 11-cisretinal initially present as rhodopsin chromophore in photoreceptor membranes. Using the isolated retina of the toad (Bufo marinus), we tested whether this procedure (‘HCHO’ method), in combination with a formaldehyde-free extraction procedure (‘i/h’ method) and the analysis of extracted retinoids by high performance liquid chromatography (HPLC), can account quantitatively for light-induced changes in retinoid levels and thus serve as an alternative to spectrophotometry for tracking the formation of all-transretinol in this intact rod preparation. Initially dark-adapted retinas were incubated in bright light or in darkness and then analysed by homogenization and extraction using the HCHO and i/h methods. Combined data obtained using the two extraction procedures indicated a near-conservation of total retinoid recovered from dark-incubated and illuminated retinas, and thus accounted for light-induced changes in retinoid levels. The HCHO procedure, employing formaldehyde, isopropanol and hexane, was similar to that described by Suzuki et al. and recovered retinaldehydes including chromophoric 11-cisretinal. The i/h procedure utilized isopropanol and hexane and, unlike the HCHO method, efficiently recovered all-transretinol. Illumination (onset at time zero) that produced an approximately exponential decline of 11-cisretinal (time constant of 24 s) led to an increase and then a gradual decline in all-transretinal. The normalized peak level of all-transretinal, representing about 0.54 of the total molar quantity of recovered retinoid, developed with illumination periods of 10–80 s. The normalized level of all-transretinol reached ≃0.3 in retinas illuminated for 1 min and, with longer illuminations (up to 30 min), exhibited an approximately exponential further growth to ≃0.9 with a time constant of 9.2 min. The results indicate the workability of the HCHO and i/h extraction procedures for tracking the in situ conversion of all-transretinal to all-transretinol, a reaction thought to be important for both operation of the retinoid visual cycle and shut-off of the phototransduction cascade.

Soldering and remelting influence on fatigue strength of cobalt-chromium alloys

Statement of problem. For laboratory production, it would be economically advisable to reuse excess cobalt-chromium alloys in manufacturing partial denture frameworks. Furthermore, discrepancies in fit, modifications, repairs, and also economic factors could lead to soldering procedure on the metal framework. Purpose. This study evaluated the influence of soldering and remelting procedures on the fatigue strength of Steldent, Dentorium, and Biosil cobalt-chromium alloys, as new alloys and in a mixture of new and previously casted alloy and when submitted or not submitted to conventional soldering procedure. Material and methods. The soldering procedure was conducted in rod specimens 1.7 mm in diameter made with new, or with a mixture of new and reused alloy ( n = 10 per group). The results were compared with those of intact rods. The fatigue strength was carried out with a cyclic testing machine simulating the insertion and the removal of partial dentures. After failure, the fracture surface was examined with a scanning electron microscope. Results. The soldered specimens revealed 1119 loading cycles before fracture. These means differed statistically from those of intact alloys, which disclosed 2733 cycles before failure ( p < 0.05). Statistical analysis indicated that the soldering procedure reduced fatigue strength of all new alloys or those in a mixture of new and previously casted alloys. It was also found that the soldering procedure may cause premature failure of a removable partial denture. Photomicrographs from fracture surfaces revealed the presence of inclusions and voids. Conversely, there were no significant differences between new and reused specimens, soldered or not ( p > 0.05), which suggested that it may be possible to reuse excess cobalt-chromium alloys without compromising the fatigue strength of the framework. (J Prosthet Dent 1997;78:146-52.)

Observations on ultrastructure of the anterior adhesive areas and other anterior glands in the monogenean, Monocotyle spiremae (Monocotylidae), from the gills of Himantura fai (Dasyatididae)

Observations with the light microscope and the scanning and transmission electron microscopes have shown that the anterior end of Monocotyle spiremae has 8 slit-like apertures on the ventrolateral margins at each side of the mouth. Gland cells located next to the pharynx produce rod-shaped secretory bodies that are conveyed in ducts that open on to the surfaces of rounded lobes inside “reservoirs” behind each ventral, slit-like aperture. Rod-shaped secretory bodies are extruded into the “reservoirs” and appear to combine and form a homogeneous secretion which may bond the ventrolateral regions of the head of the parasite to a substrate. At no stage, however, were intact rods observed outside the duct endings. Gland cells that produce an ovoid secretory body also supply the head of M. spiremae, but ducts from these open dorsal and anterior to the mouth in a region where the parasite is not known to attach. There appears to be little or no chance for the ventral rods and the dorsal ovoid secretion to mix. This is the first record of a monogenean parasite with a single type of secretion supplying the ventral surfaces of the anterior end. The rods in M. spiremae differ in some respects from the rod-shaped bodies recorded previously among gyrodactylid, dactylogyrid, capsalid and acanthocotylid monogeneans.

Further analysis of the role of spectrin repeat motifs in alpha-actinin dimer formation.

Protein constructs consisting of repeats 1-4, repeats 1-3 and repeats 2-4 of the rod domain of chicken alpha-actinin were expressed as fusion proteins in Escherichia coli. Based on the evidence of circular dichroism spectra and cooperative thermal unfolding profiles both truncated rod fragments were judged to have assumed the native structural fold. The thermal stabilities were in both cases significantly lower than that of the intact rod (repeats 1-4). Analyses by sedimentation equilibrium and velocity provided further evidence to show that fragment 1-4 is entirely dimeric in the concentration range of these experiments, resembling therefore the rod domain isolated by proteolytic digestion of native alpha-actinin. Fragment 2-4, and probably also 1-3, show concentration-dependent association, with dissociation constants, estimated by sedimentation equilibrium, in the 1-10 microM range. Thus, in confirmation of earlier work, all four repeats are required to generate a maximally stable anti-parallel dimer (Kd approximately 10 pM), suggesting the presence of binding sites in all of them to allow for aligned pairing.

Regulation of intracellular pH in salamander retinal rods.

1. We measured intracellular pH (pHi) in rods isolated from the retina of the axolotl salamander, Ambystoma mexicanum, using the fluorescent indicator 2',7'-bis(carboxyethyl)-5(and -6)-carboxyfluorescein (BCECF). 2. The light exposures associated with data acquisition had no marked effect on pHi. There was no sharp change between the value obtained from the first exposure of dark-adapted rods and subsequent readings. Increasing the acquisition frequency from 1 to 10 min-1 either had no effect, or brought about a slow acidification, which was stopped or reversed when the low frequency was restored. 3. In nominally HCO3(-)-free solution at pH 7.5, the rods had a steady-state pHi of 7.09 +/- 0.02 (n = 46) and a buffering power (beta i) of 24 +/- 1 mM (pH unit)-1 (n = 48). The buffering power was virtually constant in the pH range 6.6-8.0. In the same range, pHi dependent linearly on perfusion pH (pHo) with regression coefficients of 0.4-0.5. 4. There were no significant differences between the inner and outer segment of intact rods as regards steady-state pHi or responses to experimental treatments. 5. Recovery from an intracellular acid load imposed by sodium propionate or an NH4Cl prepulse in nominally bicarbonate-free perfusate was completely blocked by decreasing the extracellular Na+ concentration to 7 mM, and slowed by 86% by applying 1 mM amiloride. 6. Introduction of 2% CO2-13 mM HCO3- caused an alkalinization that was often preceded by a transient acidification. Steady-state pHi was on average 0.1 pH units higher than in nominally bicarbonate-free solution. The mean acid extrusion rate, calculated on the assumption that CO2-HCO3- behaves as an open system, was 19% higher (31 +/- 2 mM h-1) than in a solution buffered only by Hepes (26 +/- 2 mM h-1). 7. In the presence of CO2-HCO3-, 100 microM 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) decreased the acid extrusion rate by 20% on average. Lowering the extracellular Cl-concentration to 7 mM raised pHi, but did not significantly affect the acid extrusion rate. 8. We conclude that retinal rods regulate pHi by both Na(+)-H+ exchange and mechanism(s) involving HCO3(-)-Cl- exchange. In the present conditions, the Na(+)-H+ exchanger appears as the dominant mechanism for acid extrusion.

Dynamic, spatially nonuniform calcium regulation in frog rods exposed to light.

1. In intact rods of the bullfrog, Rana Catesbeiana, that were loaded with Fura-2 by incubation, we made high-resolution measurements of Na:Ca,K exchange currents and measured cytosolic free calcium concentrations during exposure to steps of illumination. The calcium dynamics we observed are indicative of unmanipulated rods because Fura-2 had little effect on calcium buffering within the outer segment. 2. In the dark, the total concentration of calcium within the outer segment, determined by integrating the exchange current, was near 50 microM. The free calcium concentration in darkness was 200-400 nM, indicating that > of = 99% of the internal calcium was bound to buffer molecules or equivalently sequestered. 3. During saturating illumination, the concentration of free calcium near the membrane (assayed by the exchange current) fell more rapidly than the space-averaged free calcium concentration (measured with Fura-2), but both had time courses that were best described by a sum of three exponential terms. The time constants were the same for each assay, but the weighting factors were different. 4. The relationship between the exchange current and space-averaged calcium concentration is consistent with significant concentration gradients within the outer segment resulting from high buffering power, diffusional restrictions, and the fact that all net gain and loss of calcium occurs at the membrane. The data further indicate that effective buffering, and hence calcium mobility, is not uniform within the outer segment. 5. Calcium kinetics were independent of the calcium concentration, indicating that the dominant buffers effectively have a low affinity for calcium (KD >> [Ca2+]dark free). 6. The dynamics of calcium changes and of exchange currents evoked by saturating and nonsaturating illumination are completely predictable from changes in the circulating current. Calcium and current are related by a linear transformation, indicating that calcium fluxes within the outer segment are passive and that buffers equilibrate rapidly. 7. Although calcium concentrations change slowly with respect to changes in the circulating current, both measured and calculated calcium dynamics are well correlated with changes in light adaptation. Responses to test flashes depended weakly on the detailed time course of the adapting stimulus but strongly on the free cytosolic calcium concentration at the time the test flash was delivered.

Differential membrane protein phosphorylation in bovine retinal rod outer segment disk membranes as a function of disk age.

The outer segment portion of photoreceptor rod cells is composed of a stacked array of disk membranes. Newly formed disks are found at the base of the rod outer segment (ROS) and are relatively high in membrane cholesterol. Older disks are found at the apical tip of the ROS and are low in membrane cholesterol. Disk membranes were separated based on their membrane cholesterol content and the extent of membrane protein phosphorylation determined. Light induced phosphorylation of ROS disk membrane proteins was investigated using magic angle spinning 31P NMR. When intact rod outer segment preparations were stimulated by light, in the presence of endogenously available kinases, membrane proteins located in disks at the base of the ROS were more heavily phosphorylated than those at the tip. SDS-gel electrophoresis of the phosphorylated disk membranes subpopulations identified a phosphoprotein species with a molecular weight of approximately 68-72 kDa that was more heavily phosphorylated in newly formed disks than in old disks. The identity of this phosphoprotein is presently under investigation. When the phosphorylation reaction was carried out in isolated disk membrane preparations with exogenously added co-factors and kinases, there was no preferential protein phosphorylation. Taken collectively, these results suggest that within the ROS there is a protein phosphorylation gradient that maybe indicative of co-factor or kinase heterogeneity.

Light-dependent control of calcium in intact rods of the bullfrog Rana catesbeiana.

1. Using Fura-2, we measured cytosolic free calcium concentrations in rod outer segments of the bullfrog (Rana catesbeiana) under a wide range of steady-state, adapting light intensities. We also measured rod circulating currents under the same adapting conditions. 2. Both the steady-state cytosolic free calcium concentration and the steady-state circulating current were halved by background lights that isomerized approximately 100 rhodopsin molecules per rod per second. A just-measurable reduction in calcium was evoked by a background 1/10 as bright. The steady-state calcium concentration and the steady-state circulating current were proportional to each other over the full range of intensities tested, the brightest of which suppresses the circulating current by 75% and reduces flash sensitivity by about two orders of magnitude relative to the rods' sensitivity in darkness. 3. Additional experiments, in which the Na+:Ca2+,K+ exchanger in the rod outer segment was inactivated while maintaining intracellular calcium at physiological levels, demonstrated that steady-state calcium concentrations in the outer segment are set only by the influx of calcium through the light-sensitive channels and its efflux via the Na+:Ca2+,K+ exchanger. We found no measurable light-induced release of calcium from internal stores and no evidence of any other calcium flux between the cytosol and intracellular compartments. 4. Taken together, these findings lead us to conclude that over the normal operating range of the rod, the selectivity of the cGMP-gated channels for calcium does not change as a result of background illumination. 5. Our data also suggest that a major role of calcium-dependent cGMP synthesis by guanylyl cyclase is to stabilize both the circulating current and the cytosolic free calcium concentration in darkness. This would minimize the dark noise of the rod and thereby increase the reliability with which dim stimuli can be detected in the dark-adapted state.

The cGMP-phosphodiesterase and its contribution to sensitivity regulation in retinal rods

We have used the truncated outer segment preparation to measure rod cGMP-phosphodiesterase activity, as well as its modulation by Ca2+, in darkness and in light. The basal enzyme activity in darkness was approximately 0-3 s-1, and was largely independent of Ca2+ concentration from 10 nM to 10 microM. The steady state activity elicited by a step of light (lambda = 520 nm) was strongly enhanced by Ca2+, increasing from approximately 0.005 s-1/(h nu micron-2 s-1) at 10 nM Ca2+ to approximately 0.16 s-1/h nu micron-2 s-1) at 10 microM Ca2+. Based on these measurements, as well as previous measurements on the effects of Ca2+ on rod guanylate cyclase and the cGMP-gated channel, we have calculated the step response-intensity relation for the rod cell in steady state. This relation agrees reasonably well with the relation directly measured from intact rods. We have also evaluated the relative contributions from the three Ca2+ effects to rod sensitivity. At low background light intensities, the Ca2+ modulation of the guanylate cyclase appears to be the most important for sensitivity regulation. At higher light intensities, especially above half-saturation of the response, the Ca2+ modulation of the light-stimulated phosphodiesterase shows a progressively important influence on the light response; it also extends the Weber-Fechner behavior of the cell to higher intensities. The contribution of the Ca2+ modulation of the cGMP-gated channel is slight throughout.

Ca 2+ flux in retinal rod and cone outer segments: differences in Ca 2+ selectivity of the cGMP-gated ion channels and Ca 2+ clearance rates

In intact rod and cone photoreceptors of various vertebrate species, depolarization in the dark to ≥+20 mV specifically activates the cGMP-dependent conductance in the outer segment. This activation reflects a voltage-dependent decrease in cytoplasmic Ca 2+ and the consequent activation of a Ca 2+-dependent guanylyl cyclase. The conductance activation in cones in much faster in time course and larger in extent than that in rods. Simulations of the experimental results suggest that these differences arise from differences in Ca 2+ homeostasis in the rod and cone outer segments. Direct measurements demonstrate that, indeed, the Ca 2+ permeability of the cGMP-gated channels is higher in cones than in rods. Also, as was previously known, the rate of Ca 2+ efflux from cone outer segments is higher than that in rods. Therefore, a given light-dependent change in membrane current should cause a much larger and much quicker decrease in Ca 2+ concentration in cones than in rods. The activity of every Ca 2+-dependent biochemical event in the outer segment should, hence, change more rapidly and to a larger extent in cones than in rods. We propose that these kinetic and stoichiometric differences in the function of Ca 2+-dependent processes is important in explaining the difference in the transduction signal of the two receptor types.

Association of Structural Repeats in the α-Actinin Rod Domain. Alignment of Inter-subunit Interactions

Fragments of the rod domain of chicken α-actinin, which comprises four spectrin-like repeat sequences, have been prepared by expression in Escherichia coli. Electron microscopy reveals that all products containing three or four complete repeats are rod-like. Self-association of fragments was detected by chemical cross-linking and analytical equilibrium sedimentation. The intact rod domain forms a stable dimer, which does not dissociate measureably in the accessible concentration range. Elimination of either terminal repeat (repeat 1 or repeat 4) greatly diminishes the extent of dimerisation. The fragment comprising repeats 1-3 dimerises appreciably, with an association constant estimated from the sedimentation equilibrium distribution of approximately 5|mult|10 5M −1. The fragment made up of repeats 2-4 dimerises to a small extent, but also forms aggregates at high concentrations. The results are most easily reconciled with an aligned structure for the rod domain in solution, in which repeat 1 associates with repeat 4 of the partnering chain, and repeat 2 with repeat 3, rather than with a staggered structure, in which one of the terminal repeats does not participate in dimerisation. Possible explanations for the apparent difference observed between the α-actinin rod structure in solution and in two-dimensional crystalline arrays are examined.

How Does the Retinal Rod Na-Ca+K Exchanger Regulate Cytosolic Free Ca2+?

The roles of 1) inactivation of Na-Ca+K exchange and 2) Ca2+ release from discs in regulation of cytosolic free Ca2+ were examined in intact rod outer segments (ROS) purified from bovine retinas. Measurements of cytosolic free Ca2+ (with fluo-3) were combined with Ca2+ flux measurements (45Ca) in ROS that contained about 600 microM total Ca2+. Na(+)-induced Ca2+ extrusion was measured in a Ca(2+)-free medium and did not lower cytosolic free Ca2+ to below 1 nM as expected from a coupling stoichiometry of 4Na+:(1Ca(2+) + 1K+). Instead, cytosolic free Ca2+ was rapidly (20 s) lowered from about 1300 nM to 100-150 nM, while at the same time about 35% of total ROS Ca2+ was removed. During the next 40 min cytosolic free Ca2+ remained virtually steady, but total ROS Ca2+ was reduced by a further 50% at a 100-fold lower rate than that observed for the initial fast phase. The steady cytosolic Ca2+ concentration resulted from Ca2+ release from discs and subsequent removal across the plasma membrane by Na-Ca+K exchange operating at a greatly reduced rate. Addition of the alkali cation channel ionophore gramicidin led to a persistent increase in cytosolic free Ca2+ concentration to about 400 nM, presumably caused by an increase in intracellular Na+. It is suggested that cytosolic free Ca2+ is not determined by the Na+:Ca2+ coupling ratio of the exchanger, but rather by a sensor on its cytoplasmic domain that controls inactivation of the Ca2+ extrusion mode and is sensitive to intracellular Ca2+, Na+, and K+.

The calcium feedback signal in the phototransduction cascade of vertebrate rods

Intracellular free Ca (Ca i) was measured in functionally intact rod outer segments in darkness and during light responses using the fluorescent Ca indicator Indo-dextran. In darkness, Ca i was 554 ± 25 nM (n = 28) for −85 ± 2 pA of circulating dark current (I d) and declined in saturating light to a minimum value of ∼50 nM with a time course that paralleled the fall in Na:Ca,K exchange current. During a subsaturating flash response that reduced I d by 70%, Ca i fell to a minimum of ∼325 nM and recovered incompletely to a plateau of ∼450 nM that lasted ∼15 s after full recovery of I d. During a 60 s step that caused ∼7-fold reduction in sensitivity of superimposed flash responses, Ca i reached a steady-state level of ∼252 nM.

Cation fluxes and cation channels in outer segment membranes of bovine retinal rods: contamination by antibiotics applied to cattle?

Membrane vesicles were prepared from intact rod outer segments (ROSs) isolated from bovine retinas and were examined for the presence of cation-selective conductances. We performed macroscopic flux measurements in an ensemble of ROS membrane vesicles and single-channel measurements after fusion of ROS membrane vesicles with planar bilayer membranes. Two K(+)-permeable conductances were observed, the well-established cyclic GMP (cGMP) gated channel and an apparently new K+ channel with some unusual properties. Flux and single-channel data showed that the new conductance passed K+, Rb+, and Cs+ equally well but was much less permeable to Na+, Li+ and protons. Single-channel measurements revealed a linear current-voltage relationship and three unitary conductance states of 15, 11, and 8 pS, using symmetric 150 mM KCl solutions. Measured macroscopic K+ fluxes varied considerably among different preparations, suggesting some unknown regulation of the channel; the variability appeared to arise from variation in the channel's open probability, not the unit conductance or the number of channels present. The recorded single-channel events and the selectivity data are remarkably similar to those reported for antibiotic channel-forming ionophore gramicidin. We believe that the variability in both macroscopic permeability experiments and single-channel experiments may reflect a variable contamination with gramicidin applied to the animals as the topical antibiotic V-Sporin.

Effect of hydroxylamine on the subcellular distribution of arrestin (S-antigen) in rod photoreceptors.

The immunocytochemical labeling of arrestin (S-antigen) in photoreceptors of the ovine retina was examined following incubation of the retina with hydroxylamine (NH2OH), an agent known to inhibit the phosphorylation of photoactivated rhodopsin. Intact, isolated retinas bathed in medium containing 20 mM NH2OH, or in control medium lacking NH2OH, were maintained in darkness or exposed to bright light for 3 min (dark-adapted and light-adapted conditions, respectively); further incubated in darkness for 10 min; and then fixed and prepared for cryosectioning. Cryosections were incubated with anti-S-antigen monoclonal antibody MAb A2G5; with secondary antibodies that were conjugated with horseradish peroxidase; and with either 3-amino-9-ethyl carbazole or diaminobenzidine as chromogen. Anti-arrestin labeling in cryosections was then analyzed densitometrically using a light-microscopic image processing system. In dark-adapted control retinas, labeling density of the photoreceptor outer segment (OS) layer (0.061 +/- 0.004; average +/- S.E.M.) was less than that of the inner segment (IS) layer (0.138 +/- 0.011). In light-adapted control retinas, OS labeling density (0.139 +/- 0.007) exceeded IS labeling density (0.095 +/- 0.005). Incubation with NH2OH eliminated this light-dependent increase in labeling of the OS relative to that of the IS, i.e. eliminated the increase in relative OS/IS labeling. Densities of labeling were 0.110 +/- 0.006 (OS) and 0.183 +/- 0.006 (IS) in NH2OH-treated dark-adapted retinas vs. 0.078 +/- 0.004 (OS) and 0.182 +/- 0.008 (IS) in NH2OH-treated light-adapted retinas. Anti-arrestin labeling was also examined in retinas that were exposed to 3 min or 13 min of bright light and then immediately fixed.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of intracellular cyclic GMP concentration by light and calcium in electropermeabilized rod photoreceptors.

This study examines the regulation of cGMP by illumination and by calcium during signal transduction in vertebrate retinal photoreceptor cells. We employed an electropermeabilized rod outer segment (EP-ROS) preparation which permits perfusion of low molecular weight compounds into the cytosol while retaining many of the features of physiologically competent, intact rod outer segments (ROS). When nucleotide-depleted EP-ROS were incubated with MgGTP, time- and dose-dependent increases in intracellular cGMP levels were observed. The steady state cGMP concentration in EP-ROS (0.007 mol cGMP per mol rhodopsin) approached the cGMP concentration in intact ROS. Flash illumination of EP-ROS in a 250-nM free calcium medium resulted in a transient decrease in cGMP levels; this occurred in the absence of changes in calcium concentration. The kinetics of the cGMP response to flash illumination of EP-ROS were similar to that of intact ROS. To further examine the effects of calcium on cGMP metabolism, dark-adapted EP-ROS were incubated with MgGTP containing various concentrations of calcium. We observed a twofold increase in cGMP steady state levels as the free calcium was lowered from 1 microM to 20 nM; this increase was comparable to the behavior of intact ROS. Measurements of guanylate cyclase activity in EP-ROS showed a 3.5-fold increase in activity over this range of calcium concentrations, indicating a retention of calcium regulation of guanylate cyclase in EP-ROS preparations. Flash illumination of EP-ROS in either a 50- or 250-nM free calcium medium revealed a slowing of the recovery time course at the lower calcium concentration. This observation conflicts with any hypothesis whereby a reduction in free calcium concentration hastens the recovery of cytoplasmic cGMP levels, either by stimulating guanylate cyclase activity or by inhibiting phosphodiesterase activity. We conclude that changes in the intracellular calcium concentration during visual transduction may have more complex effects on the recovery of the photoresponse than can be accounted for solely by guanylate cyclase activation.

Visual transduction in dialysed detached rod outer segments from lizard retina.

1. Properties of a new preparation for studying the physiology and biochemistry of phototransduction in retinal rods are described. Whole-cell voltage clamp was used to record the generation, maintenance and light-sensitivity of dark current in rod outer segments that had been isolated from the rest of the receptor cell by detachment at the connecting cilium. 2. Detached outer segments dialysed with standard internal solution supplemented with physiological amounts of ATP (5 mM) and GTP (1 mM) developed a standing inward dark current that was the sum of three components: approximately 91% light-sensitive current, approximately 6% Na(+)-Ca2+,K+ exchange current and approximately 3% leakage current. Light-sensitive dark current (mean amplitude approximately -63 pA) was suppressed transiently by brief flashes in an intensity-dependent manner. Light responses had the same kinetics, sensitivity and intensity-response relationship as those recorded from intact rods. 3. Dialysed outer segments differed from intact rods in that intense flashes evoked saturating responses that recovered incompletely to a plateau of reduced dark current caused by incomplete inactivation of the transduction cascade. Light sensitivity was reduced for a short time following an intense flash and then recovered despite persistent reduction of dark current. This suggests that there is no fixed relationship between dark current amplitude and light sensitivity. 4. Light-sensitive dark current faded rapidly when outer segments were not supplied with nucleotides. Outer segments dialysed with solution that contained cyclic GMP, but no ATP or GTP, supported dark current at a level that increased with [cyclic GMP]. When basal phosphodiesterase (PDE) activity is inhibited, 8 microM cyclic GMP supports a dark current of approximately 70 pA. 5. Light sensitivity decreased during recordings made with solution that contained only cyclic GMP, consistent with the inhibition of G protein activation by loss of GTP. After thorough nucleoside triphosphate depletion, however, intense illumination evoked a transient increase rather than a decrease in dark current, i.e. an inverted light response. This result suggests that isomerized rhodopsin may generate a signal that causes either inhibition of basal PDE activity or release of bound cyclic GMP. 6. Sustained Na(+)-Ca2+,K+ exchange current was recorded during steady illumination when Ca2+, but not when Mg2+, was added to the dialysis solution. Exchange current increased with the amount of added Ca2+ and saturated at approximately 18 pA when the dialysis solution contained > or = 10 mM Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)

Intracellular Ca2+ sequestration and release in intact bovine retinal rod outer segments. Role in inactivation of Na-Ca+K exchange

Intracellular Ca2+ sequestration and Ca2+ release was analyzed in intact rod outer segments (ROS) purified from bovine retinas. Ca2+ influx in Ca(2+)-depleted and fully bleached ROS was mediated exclusively by the Na-Ca+K exchanger and was measured both as a rise in cytosolic free Ca2+ with fluo 3 and as a total transmembrane Ca2+ flux with 45Ca. Ca2+ fluxes across the ROS plasma membrane were not completely reversible, in small part due to inactivation of the Ca2+ extrusion mode of the Na-Ca+K exchanger but mostly due to sequestration of cytosolic Ca2+ into the intradiskal space. Ca2+ release from the intradiskal space into the cytosol could be induced by low concentrations of the Ca2+ ionophore A23187 which gave rise to increases in cytosolic free Ca2+ by several hundred nanomolar (low A23187 concentrations did not affect the Ca2+ permeability of the ROS plasma membrane). We have used intracellular Ca2+ release induced by A23187 to examine inactivation of the rapid Ca2+ efflux mode mediated by the plasma membrane Na-Ca+K exchanger. Inactivation of Na-Ca+K exchange appeared to be dependent on intradiskal Ca2+ as it was abolished by selective Ca2+ permeabilization of the disk membrane by A23187. We discuss possible physiological roles for Ca2+ sequestration and release from ROS disks.

Identification of several rod loci and cloning of the rodD locus of Streptococcus mutans

Previous work has shown that Streptococcus mutans is normally a short rod or a sphere, depending on its environment. This paper describes two distinct genetic approaches used to identify multiple loci and isolate one locus, rodD, controlling S. mutans rod shape. The first method involved isolation of a group of rod − mutants caused by transposon Tn976 insertion, and analysis of the inactivated genes by Southern hybridization. The second method involved mutagenesis via a shotgun insertion-duplication technique, isolation of a rod − mutant, and cloning the intact rod locus, employing an integration shuttle plasmid, pVA891. These approaches have led to the identification of multiple rod loci involved in determining the rod shape of S. mutans, and also cloning of one rod locus, rodD. The cloning strategy may also be useful for cloning other streptococcal genes which cannot be detected by their expression in Escherichia coli.

Shortening of the calycal process actin cytoskeleton is correlated with myoid elongation in teleost rods

Light activates the elongation of rods within teleost retinas. Rod cell elongation is mediated by actin-dependent length changes of the myoid portion of the inner segment. The actin cytoskeleton of the inner segment consists of filament bundles, which run parallel to the long axis of the rod, from the calycal processes, through the ellipsoid and into the myoid. In isolated rod inner/outer segments (RIS-ROS), myoid elongation was found to occur in the absence of net polymerization of actin into filaments. Outgrowth of actin filaments within the myoid was counterbalanced by a shortening of actin filaments within the calycal processes. In this study, we have further examined light-activated modifications of the rod cytoskeleton using rhodamine-phalloidin to stain actin filaments within retinal cryosections as well as in isolated RIS-ROS. In RIS-ROS isolated from dark-adapted green sunfish, the phalloidin-stained calycal processes appeared as long, brush-like structures, averaging 4·2 μm in length. In light-cultured RIS-ROS populations, the calycal process actin cytoskeleton shortened from 4·2 μm to 1·7 μm. In control, dark-cultured populations, RIS-ROS that did not elongate maintained long calycal process actin cytoskeletons. However, in cases where dark-cultured RIS-ROS did elongate, despite the absence of a light stimulus, myoid elongation was accompanied by a shortening of the calycal process actin cytoskeleton, suggesting that the two events are correlated with one another. In light-adapted green sunfish and in light-cultured retinas from green sunfish and the Midas cichlid, the calycal process cytoskeleton of intact rods shortened by 40–60%. Within the two-tiered retina of green sunfish, shortening of the calycal process cytoskeleton, from 5·1 μm to 2·1–3·1 μm, was only evident in the shorter, inner tier of rods. The calycal process actin cytoskeleton did not appear to shorten within the longer, outer tier of rods; here, stained processes were short, averaging 2·3 μm in length, within dark-adapted retinas. Using scanning and transmission electron microscopy, we present evidence to suggest that the plasmalemmal surface of the calycal processes shortens along with the cytoskeletal actin core. We conclude that calycal processes of teleost rods are dynamic structures which shorten during light-activated myoid elongation.