Calcium effects on frog retinal cyclic guanosine 3’,5’- monophosphate levels and their light-initiated rate of decay

When retinal sections were isolated from dark-adapted bullfrogs and placed in normal ringer’s solution, they contained 40.7 +/- 0.2 pmol cGMP/mg protein (mean +/- SEM, 30 samples). When isolated, dark-adapted retinal sections were removed from normal ringer’s solution and placed in calcium-deficient ringer’s solution with 3 mM EGTA, there was about a threefold rise in cyclic GMP (cGMP) levels by 1.5 min and about a 10-fold rise by 5 min. The cGMP level remained high with no detectable decrease for at least 40 min (the longest time measured). When isolated, dark- adapted retinal sections were removed from normal ringer’s solution and placed in ringer’s solution which contained high- calcium (20 mM CaCl(2)), there was a slow but significant decrease in cGMP levels. After 20 min in high-calcium ringer’s solution the cGMP level was 0.58 +/- 0.07 (mean +/- SEM, eight samples) of the cGMP level in normal ringer’s solution incubated for the same time. The rate at which 10-fold elevated cGMP levels in low calcium decreased upon illumination was examined using quick-freezing techniques on the retinal sections. The elevated cGMP level in retinal sections incubated in low-calcium decreased upon illumination was examined using quick-freezing techniques on the retinal sections. The elevated cGMP level in retinal sections incubated in low-calcium ringer’s solution was found to decay about 15-fold faster than cGMP levels in retinal sections incubated in normal ringer’s solution. The CGMP level in low calcium was significantly different (P=0.005) after 1 s illumination, whereas the cGMP level in normal calcium was not significantly different.     

Magnitude of increase in retinal cGMP metabolic flux determined by 18O incorporation into nucleotide alpha-phosphoryls corresponds with intensity of photic stimulation

The property of cyclic nucleotide phosphodiesterases to catalyze 3'-P--O bond cleavage and the insertion of a single nonexchangeable atom of 18O from [18O]water into the phosphoryl of the 5'-nucleotide product has been utilized as a means for measuring the hydrolytic flux of cGMP and cAMP in isolated dark-adapted intact rabbit retinas. Without illumination 18O labeling of guanine nucleotide (GTP and GDP) alpha-phosphoryls proceeds linearly for at least 80 s at a rate of 3.3 nmol of 18O/s.g of retina (wet weight). This rate is estimated to be approximately 8 times greater in the rod outer segment layer where over 90% of retinal cGMP metabolic components reside. Photic stimulation during a 20-s incubation was provided by intermittent flashes of light representing 800 ms of total illumination. Light stimuli over a range of intensities of greater than 3 log units commencing with a minimally detectable intensity produce graded increments in the rate of 18O incorporation into guanine nucleotide alpha-phosphoryls to a maximum increase of 5-fold. On the basis of only the 800-ms period of illumination this maximum increase is 125-fold. Steady state levels of retinal cGMP are not altered appreciably over this greater than 3 log range of light intensities but a light stimulus exceeding this intensity range causes an approximate 50% decrease in retinal cGMP concentration and a relative decline in the maximal rate of 18O labeling of guanine nucleotide alpha-phosphoryls. No light-related increases were detected in 18O incorporation into adenine nucleotide alpha-phosphoryls nor the gamma-phosphoryls of GTP or ATP or Pi. These observations indicate that light stimuli over greater than 3 log of light intensity produce incremental increases in cGMP metabolic flux that result from comparable increases in the rates of both cGMP generation and cGMP hydrolysis. It is postulated that increases in cGMP metabolic flux rather than changes in cGMP steady state levels are integral to phototransduction by a mechanism that involves the coupling of cGMP synthesis and/or hydrolysis to either the release of calcium from disc membranes or the inhibition of Na+ conductance by the photoreceptor membrane. This is suggested to occur by an energy-linked process and/or the generation of protons.     

Light-induced changes of cyclic GMP content in frog retinal rod outer segments measured with rapid freezing and microdissection

Cyclic GMP concentration was measured in the rod outer segments (ROS) of the isolated frog retinas. Retinas were quickly frozen in 0.5 s after the short light flash producing 90%-saturated late receptor potential (2,000 rhodopsins bleached per rod). ROS were obtained by microdissection, the cGMP levels were determined by radioimmunoassay method. No detectable changes in cGMP concentration was found in this stimulus condition. Dark-adapted ROS contained 46.3 +/- 2 pmol/mg. 3-s bright illumination (ca. 10(7) rhodopsins bleached per rod per second) led to approximately 30% drop in cGMP content. It is supposed that the main part of cGMP with the ROS is in the bound state and therefore fast light-induced changes in its minor free fraction may escape the detection.     

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.     

Oxygen distribution and consumption in the cat retina during normoxia and hypoxemia

Oxygen tension (PO2) was measured with microelectrodes within the retina of anesthetized cats during normoxia and hypoxemia (i.e., systemic hypoxia), and photoreceptor oxygen consumption was determined by fitting PO2 measurements to a model of steady-state oxygen diffusion and consumption. Choroidal PO2 fell linearly during hypoxemia, about 0.64 mmHg/mmHg decrease in arterial PO2 (PaO2). The choroidal circulation provided approximately 91% of the photoreceptors' oxygen supply under dark-adapted conditions during both normoxia and hypoxemia. In light adaptation the choroid supplied all of the oxygen during normoxia, but at PaO2's less than 60 mmHg the retinal circulation supplied approximately 10% of the oxygen. In the dark-adapted retina the decrease in choroidal PO2 caused a large decrease in photoreceptor oxygen consumption, from approximately 5.1 ml O2/100 g.min during normoxia to 2.6 ml O2/100 g.min at a PaO2 of 50 mmHg. When the retina was adapted to a rod saturating background, normoxic oxygen consumption was approximately 33% of the dark-adapted value, and hypoxemia caused almost no change in oxygen consumption. This difference in metabolic effects of hypoxemia in light and dark explains why the standing potential of the eye and retinal extracellular potassium concentration were previously found to be more affected by hypoxemia in darkness. Frequency histograms of intraretinal PO2 were used to characterize the oxygenation of the vascularized inner half of the retina, where the oxygen distribution is heterogeneous and simple diffusion models cannot be used. Inner retinal PO2 during normoxia was relatively low: 18 +/- 12 mmHg (mean and SD; n = 8,328 values from 36 profiles) in dark adaptation, and significantly lower, 13 +/- 6 mmHg (n = 4,349 values from 19 profiles) in light adaptation. Even in the dark-adapted retina, 30% of the values were less than 10 mmHg. The mean PO2 in the inner (i.e., proximal) half of the retina was well regulated during hypoxemia. In dark adaptation it was significantly reduced only at PaO2's less than 45 mmHg, and it was reduced less at these PaO2's in light adaptation.     

Calcium and cyclic nucleotide regulation in incubated mouse retinas.

When retinas from dark-adapted C57BL/6 mice were incubated in the dark for 5 min at 37 degrees C in Earle's medium, they contained 80-120 pmol/mg protein of cGMP and about 13 pmol/mg protein of cAMP. When the incubation in darkness was in calcium-deficient Earle's medium with 3 mM EGTA, a 10-20 fold increase occurred in the cGMP level, peaking at 2-3 min, but no change occurred in cAMP. This elevated level fell in 3 min to normal dark levels on return to normal Earle's medium, but was still about three times that of control levels after 15 min in EGTA-containing solution. Bright light after 2 min of dark incubation of dark-adapted retinas resulted in a 40-50% fall in cGMP, and bright light sharply reduced the elevated dark cGMP level of retinas in calcium-deficient media with 3 mM EDTA. However, no depression of normal dark levels of cGMP has thus far been obtained by increasing external calcium levels, even in the presence of the ionophore A23187. All the above phenomena involving dark cGMP levels and calcium are similar in Earle's medium with 100 mM of K+ substituted for Na+. Congenic rodless (rd/rd) mouse retinas have less than 5% of control cGMP and show only traces of calcium sensitivity. Thus, the above phenomena in controls are likely to be largely occurring in rods. The data suggest a dependency of the dark cGMP level on the calcium level, but that the light-induced fall in cGMP may largely be calcium insensitive.     

Light-induced decreases in cGMP concentration precede changes in membrane permeability in frog rod photoreceptors

This study examines whether changes in cGMP concentration initiated by illumination of frog rod photoreceptors occur rapidly enough to implicate cGMP as an intermediate between rhodopsin activation in the disc membrane and permeability changes in the plasma membrane. Previous studies using whole retinas or isolated outer segments have provided conflicting evidence on the role of cGMP in the initial events of phototransduction. The rod photoreceptor preparation employed in this work consists of purified suspensions of outer segments still attached to the mitochondria-rich ellipsoid portion of the inner segment. These photoreceptors are known to retain normal electrophysiological responses to illumination and have cGMP levels comparable to those measured in the intact retina. When examined under several different conditions, changes in cGMP concentrations were found to occur as rapidly or more rapidly than the suppression of the membrane dark current. Subsecond changes in cGMP concentration were analyzed with a rapid quench apparatus and confirmed by comparison with a rapid freezing technique. In a 1 mM Ca2+ Ringer's solution, cGMP levels decrease to 65% of their final extent within 200 ms after bright illumination; changes in membrane dark current follow a similar time course. When the light intensity is decreased to 8000 rhodopsins bleached per rod per s, the light-induced cGMP decrease is completed within 50 ms, with 7 X 10(5) cGMP molecules hydrolyzed per rhodopsin bleached. During this time the dark current has not yet begun to change. Thus, under physiological conditions it is clear that changes in cGMP concentration precede permeability changes at the plasma membrane. The correlation of rapid changes in cGMP levels with changes in membrane current leave open the possibility that changes in cGMP concentration may be an obligatory step in the reaction sequence linking rhodopsin activation by light and the resultant decrease in sodium permeability of the plasma membrane.     

Light-induced changes in energy metabolites, guanine nucleotides, and guanylate cyclase within frog retinal layers

Freeze-dried sections were prepared from retinas of frogs which were dark-adapted or exposed to varying periods of light. Samples of the discrete layers were dissected, weighed, and analyzed for energy metabolites, guanylate compounds, and the enzyme guanylate cyclase. ATP and P-creatine were measured in both dark- and light-adapted retinas. There was a gradient in ATP and P-creatine levels in dark-adapted retinas, with the lower concentrations in the photoreceptors, and increasing concentrations in the inner retina. After light adaptation, concentrations increased, an observation which supports the concept that transmitter release occurs in the dark and ceases in the light. The sum of GTP plus GDP, GDP, and cyclic GMP were analyzed in dark-adapted retinas and after exposure to 2 min or 2 h of room light. GDP was rather uniformly distributed in the retinal layers, was increased by 2 min of light in all layers but the outer nuclear, and remained elevated at 2 h in the inner retina. GTP values showed a marked localization in the outer nuclear layer, which increased after 2 min or 2 h of illumination; in all other layers GTP was decreased by light. Cyclic GMP in the dark was highest in the photoreceptor cells, decreasing to one-third after 2 min of light; there were significant increases in the outer plexiform and inner nuclear layers at this time. Cyclic GMP remained low in the photoreceptor cells even after 2 h of light, while the inner layers returned to dark values. Guanylate cyclase, like cyclic GMP, was largely confined to the photoreceptor cells and showed a maximal increase after 2 min of light exposure.     

Effects of light and darkness on oxygen distribution and consumption in the cat retina

These experiments were done to investigate the effects of light and darkness on the oxygenation of the retina in anesthetized cats. Measurements were made with double-barreled oxygen microelectrodes capable of recording both oxygen tension (PO2) and local voltages. Diffuse white illumination presented to a dark-adapted retina led to an increase in PO2 of up to 30 mmHg in the outer half of the retina. Changes were maximal at approximately 75% depth, corresponding to the outer nuclear layer. No change or decrease in PO2 was observed in the inner retina. Light-evoked increases in outer retinal PO2 were graded with the duration and strength of illumination, and were maximal in response to 60 s of illumination at rod saturation. For these stimuli, the increase at the onset of illumination was slower (average half-time, 12.2 s) than the recovery at the end of illumination (average half-time, 5.9 s), but for stimuli above rod saturation, PO2 recovered much more slowly. The profile of PO2 was measured during electrode penetration and withdrawal and during light and dark adaptation. Dark-adapted profiles were characterized by a minimum PO2 of nearly 0 mmHg at depths of 65-85%, and a steep gradient from the minimum to the choroid. During light adaptation at rod saturation, PO2 was elevated in the outer half of the retina and the minimum was eliminated. Fits of the profiles to a one-dimensional model of oxygen diffusion indicated that light reduced the oxygen consumption of the outer retina to approximately 50% of its dark-adapted value.     

Electrical and adaptive properties of rod photoreceptors in bufo marinus. II. Effects of cyclic nucleotides and protaglandins

Substances known to alter cyclic nucleotide levels in cells were applied to the isolated toad retina and effects on rod electrical and adaptive behavior were studied. The retina was continually superfused in control ringer’s or ringer’s containing one or a combination of drugs, and rod activity was recorded intracellularly. Superfusion with cGMP, Bu(2)GMP, isobutylmethylxanthine (IBMX; a phosphodiesterase inhibitor), or PGF(2α) (a prostaglandin) caused effects in rods that closely match those observed when extracellular Ca(2+) levels were lowered. For example, short exposures (up to 6 min) of the retina to these substances caused depolarization of the membrane potential, increase in response amplitudes, and some changes in waveform; but under dark-adapted or partially light-adapted conditions receptor sensitivity was virtually unaffected. That is, the position of the V-log I curve on the intensity axis was determined by the prevailing light level, not by drug level. These drugs, like lowered extracellular Ca(2+), also decreased the period of receptor saturation after a bright-adapting flash, resulting in an acceleration of the onset of membrane and sensitivity recovery during dark adaptation.

Long-term (6-15 min) exposure of a dark-adapted retina to 5 mM IBMX or a combination of IBMX and cGMP caused a loss of response amplitude and a desensitization of the rods that was similar to that observed in rods after a long-term low Ca(2+) (10(-9)M) treatment. Application of high (3.2 mM) Ca(2+) to the retina blocked the effects of applied Bu(2)cGMP. PGE(1) superfusion mimicked the effects of increasing extracellular Ca(2+). The results show that increased cGMP and lowered Ca(2+) produce similar alterations in the electrical activity of rods. These findings suggest that Ca(2+) and cGMP are interrelated messengers. We speculate that low Ca(2+) may lead to increased intracellular cGMP, and/or that applied cGMP, and/or that applied cGMP may lower cytosol Ca(2+), perhaps by stimulating Ca(2+)- ATPase pumps in the outer segment.

     

Effect of light and calcium on cyclic GMP synthesis in rod outer segments of toad retina

The rod outer segments of toad retina contain a guanylate cyclase activity of about 3 +/- 1 nmol of cGMP formed/min per mg protein. In darkness this value is largely independent of the Ca2+ concentration, although it is enhanced by light upon lowering the Ca2+ concentration from 10(-5) to 10(-8) M. The activating effect of light on cyclase at low Ca2+ concentrations is enlarged upon increasing the light intensity. With a flash of light bleaching 7 X 10(-2) percent of rhodopsin, cyclase activity increased by a factor of 30 when Ca2+ levels dropped from 10(-5) to 10(-8) M. In view of recent observations that shortly after a flash of light the calcium activity inside the photoreceptor cell decreases, it seems likely that Ca2+ plays a regulatory role on cGMP metabolism in visual excitation.     

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.     

Effects of melatonin and 6-methoxy-tetrahydro-beta-carboline in light induced retinal damage: a computerized morphometric method

The effects of melatonin and a related 5-methoxy-indole, 6-methoxy-1,2,3,4-tetrahydro-beta-carboline (6-MeO-THBC) were investigated in rats on the development of retinal degeneration in presence of high intensity illumination (HII). A morphometric method is used in which the degree of degeneration was evaluated by a computer-coupled graphical analyzer. Instead of measuring individual thicknesses of different retinal layers at various loci we measured large areas of retinal light microscopic sections. Thus the influence of sporadic artefactual and other fluctuations in the thickness of various layers of the retina can be essentially reduced. Continuous light produced significant degeneration of the retina and the degree of degeneration was further increased by both studied compounds and even more by 6-MeO-THBC. The role of melatonin and 6-MeO-THBC in retinal physiology is discussed.     

Guanylate cyclase in rod outer segments of the toad retina. Effect of light and Ca2+

Guanylate cyclase activity was measured in disrupted rod outer segments of the toad retina. The experiments showed that cGMP is synthesized from GTP at a rate of 3 +/- 1 nmol/min per mg protein. In darkness this value is largely independent of the Ca2+ concentration, while it is enhanced by flashes of light of increasing intensity upon lowering Ca from 10-5 to 10-8 M. In view of recent observations that shortly after a flash of light calcium activity inside the photoreceptor cell decreases, it seems likely that calcium plays a regulatory role in cGMP metabolism in visual excitation.     

Detection of a light-sensitive pool of cGMP in goldfish cone color receptors by immunocytochemistry

A procedure for the immunocytochemical localization of cGMP in the goldfish retina is described. The procedure is then used to evaluate the presence of cGMP in cone color receptors in retinas from dark-adapted and light stimulate fish. The results show that cone color receptors in dark-adapted retinas contain large amounts of immunocytochemically defined cGMP in the ellipsoid region, which are greatly reduced after light exposure.     

Light-induced losses and dark recovery rates of guanosine 3'',5''-cyclic monophosphate in rod outer segments of intact amphibian photoreceptors

We used an apparatus in which pieces of dark-adapted amphibian retinas (Rana pipiens, Bufo marinus) obtained under infrared illumination were exposed to precise intervals of 500-nm illuminations, and then frozen by contact of their outer segment surface with a liquid helium-cooled copper mirror. Sections of the frozen outer segment layer were obtained in a cryostat and then assayed for total extractable cyclic 3',5'-guanosine monophosphate (cGMP). Significant losses of cGMP with respect to the dark level were evident as early as 60 ms after light onset. With dim subsecond illuminations these losses were surprisingly large, which suggests a previously underestimated magnification in the cGMP cascade, or a transient substantial inhibition of guanylate cyclase activity in combination with increased cyclic GMP phosphodiesterase activity. Within the subsecond period, significant losses that were proportional to light intensity (2-log-unit range) and duration (60-550 ms) were generally not evident. However, losses significantly proportional to these factors became evident with durations of 1 s or longer. When pieces of retina were first illuminated (10 or 60 ms), then held in darkness for increasing periods before freezing, we observed a continuous loss of cGMP during the early postillumination dark period, followed by a recovery of the total cGMP level. The times for recovery to the preillumination level appear to be significantly longer than times reported for the recovery of the photoreceptor membrane potential after similar light exposures.     

Near-ultraviolet radiation suppresses melatonin synthesis in the chicken retina: a role of dopamine

Effects of near-ultraviolet radiation (UV-A; 325-390 nm, peak at 365 nm) on melatonin content and activity of serotonin N-acetyltransferase (AA-NAT; a key regulatory enzyme in melatonin biosynthesis) were examined in the retina of chickens. Acute exposure of dark-adapted animals to UV-A light produced a marked decline in melatonin content and AA-NAT activity of the retina. The magnitude of the observed changes was dependent upon duration of the light pulse and age of chickens, with 1-2-week old birds being more sensitive to UV-A action than 6-7-week old ones. The decrease in the nocturnal AA-NAT activity evoked by a 5-min UV-A pulse gradually deepened during the first 30 min after the return of chickens to constant darkness, then the enzyme activity began to rise, reaching nearly complete restoration within 2.5 hr. Systemic administration to chickens of alpha-methyl-p-tyrosine (an inhibitor of catecholamine synthesis; 0.3 g/kg) blocked the suppressive effect of UV-A light on retinal AA-NAT activity. Haloperidol, sulpiride (blockers of D2-family of dopamine (DA) receptors) and 2-chloro-11-(4-methylpiperazino)dibenz[b,f]oxepin (an antagonist of D4-DA receptors), given intraocularly (1-100 nmol/eye) prevented the UV-A light-evoked decrease in AA-NAT activity in the chicken retina in a dose-dependent manner, while raclopride (300 nmol/eye), an antagonist of D2/D3-DA receptors, was ineffective. In dark-adapted chickens exposure to UV-A light increased the DA content of the retina. It is concluded that UV-A radiation, similar to visible light, potently suppresses melatonin biosynthesis in the retina of chicken, with a D4-dopaminergic signal playing the role of an intermediate in this action.     

Oxygen distribution and consumption in the macaque retina

The oxygen distribution in the retina of six anesthetized macaques was investigated as a model for retinal oxygenation in the human retina in and adjacent to the fovea. P(O2) was measured as a function of retinal depth under normal physiological conditions in light and dark adaptation with O(2) microelectrodes. Oxygen consumption (Q(O2)) of the photoreceptors was extracted by fitting a steady-state diffusion model to P(O2) measurements. In the perifovea, the P(O2) was 48 +/- 13 mmHg (mean and SD) at the choroid and fell to a minimum of 3.8 +/- 1.9 mmHg around the photoreceptor inner segments in dark adaptation, rising again toward the inner retina. The P(O2) in the inner half of the retina in darkness was 17.9 +/- 7.8 mmHg. When averaged over the outer retina, photoreceptor Q(O2) (called Q(av)) was 4.6 +/- 2.3 ml O(2).100 g(-1).min(-1) under dark-adapted conditions. Illumination sufficient to saturate the rods reduced Q(av) to 72 +/- 11% of the dark-adapted value. Both perifoveal and foveal photoreceptors received most of their O(2) from the choroidal circulation. While foveal photoreceptors have more mitochondria, the Q(O2) of photoreceptors in the fovea was 68% of that in the perifovea. Oxygenation in macaque retina was similar to that previously found in cats and other mammals, reinforcing the relevance of nonprimate animal models for the study of retinal oxygenation, but there was a smaller reduction in Q(O2) with light than observed in cats, which may have implications for understanding the influence of light under some clinical conditions.     

Inhibitory modulation of photoreceptor melatonin synthesis via a nitric oxide-mediated mechanism

Nitric oxide (NO) has been suggested to have many physiological functions in the vertebrate retina, including a role in light-adaptive processes. The aim of this study was to examine the influence of the NO-donor sodium nitroprusside (SNP) on the activity of arylalkylamine-N-acetyltransferase (AA-NAT; EC. 2.3.1.87), the activity of which responds to light and reflects the changes in retinal melatonin synthesis—a key feature of light-adaptive responses in photoreceptors. Incubation of dark-adapted and dark-maintained retinas with SNP lead to the NO-specific suppression of AA-NAT activity, with NO suppressing AA-NAT activity to a level similar to that seen in the presence of dopaminergic agonists or light. Increased levels of cGMP appeared to be causally involved in the suppression of AA-NAT activity by SNP, as non-hydrolysable analogues of cGMP and the cGMP-specific phosphodiesterase (PDE) inhibitor zaprinast also significantly suppressed AA-NAT activity, while an inhibitor of soluble guanylate cyclase blocked the effect of SNP. While this chain of events may not be part of the normal physiology of the retina, it could be important in pathological circumstances that are associated with marked increase in levels of cGMP, as is found to be the case in certain forms photoreceptor degeneration, which are produced by defects in cGMP phosphodiesterase activity.     

Light-induced changes of cyclic GMP content in frog retinal rod outer segments measured with rapid freezing and microdissection

Cyclic GMP concentration was measured in the rod outer segments (ROS) of the isolated frog retinas. Retinas were quickly frozen in 0.5 s after the short light flash producing 90%-saturated late receptor potential (2,000 rhodopsins bleached per rod). ROS were obtained by microdissection, and cGMP levels were determined by radioimmunoassay method. No detectable changes in cGMP concentration was found in this stimulus condition. Dark-adapted ROS contained 46.3 ± 1.5 pmole cGMP per mg dry weight, flash-illuminated ones –45 ± 2 pmole/mg. 3-s bright illumination (ca. 10⁷ rhodopsins bleached per rod per second) led to approximately 30% drop in cGMP content. It is supposed that the main part of cGMP within the ROS is in the bound state and therefore fast light-induced changes in its minor free fraction may escape the detection.