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.     

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-initiated changes of cyclic guanosine monophosphate levels in the frog retina measured with quick-freezing techniques

Although there is good agreement that light reduces the amount of cyclic GMP (cGMP) in the retina, the exact time-course of this decrease is not well established. Bullfrog retinal sections were isolated under infrared light and quick-frozen with liquid nitrogen-cooled, metal hammers after exposure to various intensities of continuous illumination. This quick-freezing should stop the degradation of cGMP within 50-100 ms. The frozen retinal sections were then slowly warmed up in the presence of perchloric acid to denature enzymes involved in cGmp metabolism. cGMP was determined by radioimmunoassay and comparison was made between light- and dark-adapted retinal sections from the same animal. The average cGMP concentration was 44.3 +/- 0.7 pmol cGMP/mg protein or 170.9 +/- 3.2 pmol cGMP/retina. After 1 s of illumination no significant change in cGMP concentration was found even with the brightest light used (approximately 7 x 10(7) rhodopsins bleached/second per rod. At this intensity the first significant decrease in cGMP from dark-adapted levels was detected 3-5 s after the initiation of illumination; cGMP decayed to 70-75% of the dark-adapted value after approximately 30 s. With lower intensity illumination the cGMP levels recovered to dark-adapted levels after the initial decrease even though the bleaching light remained on.     

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.     

Interaction between the main components of the vertebrate retinal rod phototransduction system in solutions of detergent n-nonyl-β-D-glucoside

cGMP-Specific phosphodiesterase (PDE6) is the key enzyme of the phototransduction system of vertebrate retinal rod outer segments (ROS). The properties of PDE in extracts prepared by solubilization of bovine ROS in a high concentration (0.5% w/v) of detergent n-nonyl-β-D-glucoside (NG) and following centrifugation (ROS-NG) have been studied. Basal PDE activity of the preparations was low, but it greatly (>50-fold) increased (up to ∼20 μmol cGMP hydrolyzed/min per mg rhodopsin (R)) in the presence of trypsin. In bleached GTPγS-containing preparations the specific PDE activity was dependent on ROS-NG concentration and was half-maximal at about 0.8 μM of ROS G protein transducin (Gt). In dark-adapted GTPγS-containing ROS-NG preparations bleaching of 0.2% of the rhodopsin resulted in half-maximal PDE activation. The same result was obtained when PDE in dark-adapted ROS-NG preparations was activated by addition of a highly purified bleached rhodopsin solubilized by 0.5% solution of NG. The results demonstrate that the presence of NG has no significant influence either on the properties of the main ROS phototrans-duction system elements (R, Gt and PDE) or on the interaction between photoactivated R and Gt and suggest that the detergent NG can be used for crystallization of the rhodopsin-transducin complex.     

Changes in cGMP concentration correlate with some, but not all, aspects of the light-regulated conductance of frog rod photoreceptors

Cyclic GMP has been implicated in controlling the light-regulated conductance of rod photoreceptors of the vertebrate retina. However, there is little direct evidence correlating changes in cGMP concentration with the light-regulated permeability mechanism in living cells. A preparation of intact frog rod outer segments suspended in a Ringer's medium containing low Ca2+ has been used to demonstrate that initial changes in total cellular cGMP concentration parallel changes in the light-regulated membrane current over a wide range of light intensities. At light intensities bleaching from 160 to 5.6 X 10(6) rhodopsin molecules/rod/s, decreases in the response latency for the cGMP kinetics parallel decreases in the latent period of the electrical response. Further, changes in the rate of the cGMP decrease parallel the rate of membrane current suppression as the light intensity is varied. Up to 10(5) cGMP molecules are hydrolyzed per photolyzed rhodopsin, consistent with in vitro studies showing that each bleached rhodopsin can activate over 100 phosphodiesterase molecules. Addition of the Ca2+ ionophore, A23187, does not affect the initial kinetics of the cGMP decrease or of the electrical response, excluding a direct role for Ca2+ in the initial events of phototransduction. These results are consistent with cGMP being the intracellular messenger that links rhodopsin isomerization with changes in membrane permeability upon illumination. It is unlikely, however, that light-induced changes in total cGMP concentration are the sole regulators of membrane current. This is suggested by several observations: at bright light intensities, the subsecond light-induced cGMP decrease is essentially complete prior to complete suppression of membrane current; maximal light-induced decreases in cGMP concentration occur at all light intensities tested, whereas the extent of membrane current suppression varies over the same range of light intensities; changing the external Ca2+ concentration from 1 mM to 10 nM in the dark causes an increase in membrane current that is significantly more rapid than corresponding changes in cGMP concentration. Thus, light-induced changes in total cellular cGMP concentration correlate with some, but not all, aspects of the visual excitation process in vertebrate photoreceptors.     

Light-induced conformational changes in the extradiscal regions of bovine rhodopsin

Light-induced conformational changes occurring at the cytosolic surface of rhodopsin were investigated by performing limited digestions of native and illuminated visual pigment with thermolysin, Arg-C endoproteinase, papain and proteinase K. A higher susceptibility of the extradiscal regions of the bleached pigment to the proteases were observed together with altered capacities of the digested bleached rhodopsins to activate the cGMP phosphodiesterase. The overall results strongly suggest that light induces conformational changes not only in the C-terminal end but also in the second and the third extradiscal loop of rhodopsin.     

Regulation by light of cyclic nucleotide-dependent protein kinases and their substrates in frog rod outer segments

Cyclic nucleotides (both cAMP and cGMP) stimulate the phosphorylation of several proteins of 65-70, 50-52, 21, 13, and 12 kD in rod outer segments (ROS) of the frog retina. Subcellular fractionation showed that phosphopeptides of 67, 21, 13, and 12 kD were soluble and phosphopeptides of 69, 67, 50-52, and 12 kD were membrane associated at physiological ionic strength. Components I and II, 13 and 12 kD, respectively, are the major cyclic nucleotide-dependent phosphoproteins of ROS and have been reported to be phosphorylated in the dark and dephosphorylated in the light. Under unstimulated conditions, phosphorylated Components I and II were found in the soluble fraction. Cyclic nucleotide stimulation of phosphorylation resulted in increased phospho-Components I and II in the soluble fraction, and phospho-Component II on the membrane. Light had no effect on the phosphorylation level of soluble Components I and II, but it caused a depletion within 1 s of the membrane-bound phospho-Component II. A half-maximal decrease in membrane-bound Component II was seen at 5 x 10(5) rhodopsins bleached per outer segment. The cyclic nucleotide-dependent protein kinase(s) were found primarily in the peripheral membrane fraction of ROS proteins. 8-bromo cyclic AMP was two orders of magnitude more effective than 8-bromo cyclic GMP at stimulating Component I and II phosphorylation. An active peptide of the Walsh inhibitor of cAMP-dependent protein kinase [PKI(5-22)amide] blocked the phosphorylation with an IC50 of 10 nM. Photoaffinity labeling studies with 8-N3-cAMP and 8-N3-cGMP revealed the presence of a 52-kD band specifically labeled with 8-N3-cAMP, but no specific 8-N3-cGMP labeling. These data suggest that cyclic nucleotide-dependent protein phosphorylation in ROS occurs via the activation of a cAMP-dependent protein kinase.     

Transducin GTPase provides for rapid quenching of the cGMP cascade in rod outer segments

The role of transducin GTPase in rapid cGMP phosphodiesterase quenching was studied by simultaneous registration of GTP hydrolysis and phosphodiesterase activity in the same rod outer segments (ROS) preparation. The results thus obtained allow the conclusion that: (i) phosphodiesterase quenching coincides with transducin-bound GTP hydrolysis independently of ROS concentration; (ii) an increase in the ROS concentration results in the acceleration of cascade quenching due to the existence of a GTPase accelerating mechanism in ROS; (iii) approximation to physiological conditions (protein concentration, temperature) provides a transducin GTPase rate equal to 1–2 turnovers per second i.e., sufficiently high for satisfying the real rate of photoresponse reversion in dark-adapted rods.     

8-Br-cGMP和cGMP对蟾蜍视杆光敏感通道作用的比较研究

在蟾蜍视网膜的视杆外段上进行片膜钳记录.在同一片膜中,8-Br-cGMP开启的通道,似乎是cGMP所开启的同一光敏感通道.在相同的片膜电位下,由同一片膜诱发同样大小的膜电流,所灌流的8-Br-cGMP溶液的浓度要大大低于cGMP溶液的浓度.再者,8-Br-cGMP被磷酸二酯酸水解的速率,比cGMP的要慢得多.结果表明,在开启视杆外段的光敏感通道的过程中,cGMP分子中的3＇,5＇-环一磷酸可能起主要作用.     

cGMP suppresses GTPase activity of a portion of transducin equimolar to phosphodiesterase in frog rod outer segments. Light-induced cGMP decreases as a putative feedback mechanism of the photoresponse.

In rod photoreceptor cells, the light response is triggered by an enzymatic cascade that causes cGMP levels to fall: excited rhodopsin (Rho*)----rod G-protein (transducin, Gt)----cGMP-phosphodiesterase (PDE). This results in the closure of plasma membrane channels that are gated by cGMP. PDE activation by Gt occurs when GDP bound to the alpha-subunit of Gt (Gt alpha) is exchanged with free GTP. The interaction of Gt alpha-GTP with the gamma-subunits of PDE releases their inhibitory action and causes cGMP hydrolysis. Inactivation is thought to be caused by subsequent hydrolysis of Gt alpha-GTP by an intrinsic Gt-GTPase activity. Here we report that there are two portions of Gt in frog rod outer segments (ROS) expressing different rates of GTP hydrolysis: 19.5 +/- 3 mmol of Gt/mol of Rho, equivalent to that amount which participates in PDE activation, hydrolyzing GTP at a rate of approximately 0.6 turnover/s ("fast") and the remaining Gt (80.5 +/- 3 mmol/mol Rho) hydrolyzing GTP at a rate of 0.058 +/- 0.009 turnover/s. Fast GTPase activity is abolished in the presence of cGMP. This effect occurs over the physiological range of cGMP concentration changes in ROS, half-saturating at approximately 2 microM and saturating at 5 microM cGMP. cGMP-dependent suppression of GTPase is specific for cGMP; cAMP in millimolar concentration does not affect GTPase, while the poorly hydrolyzable cGMP analogue, 8-bromo-cGMP, mimics the effect. GTPase regulation by cGMP is not affected by Ca2+ over the concentration range 5-500 nM, which spans the physiological changes in cytoplasmic Ca2+ in rod cells. We suggest that the fast cGMP-sensitive GTPase activity is a property of the Gt that activates PDE. In this model, cGMP serves not only as a messenger of excitation but also modulates GTPase activity, thereby mediating negative feedback regulation of the pathway via PDE turnoff: a light-dependent decrease in cGMP accelerates the hydrolysis of GTP bound to Gt, resulting in the rapid inactivation of PDE.     

Activation of bovine rod outer segment phospholipase C by arrestin.

Phospholipase C (PLC) enzyme activity in rod outer segment (ROS) membranes bleached in the presence of ATP and GTP was assayed using exogenously added [3H]phosphatidylinositol 4,5-bisphosphate vesicles as substrate. The addition of the soluble ROS protein arrestin (also known as S-antigen or 48K protein) to ROS membranes activated PLC 2-3.4-fold. This activation was dose-dependent, and maximal activation was observed at an arrestin concentration of congruent to 110-220 nM. PLC activation by arrestin was dependent on ROS protein concentration and free Ca2+. Soluble PLC (s-PLC) enzyme activity present in hypotonic extracts of bleached ROS was also activated 2-4-fold by arrestin. Maximum activation of s-PLC by arrestin was observed at free Ca2+ of 80 nM. Arrestin activation of s-PLC was not affected by urea-treated and extensively washed ROS membranes, suggesting that rhodopsin was not required for the observed effect of arrestin on s-PLC. The results are indicative of a direct interaction of arrestin with s-PLC, resulting in the activation of the latter. Based on these results and the documented binding of arrestin to bleached and phosphorylated rhodopsin, a model for the light activation of PLC in ROS is proposed.     

Inactivation of photoexcited rhodopsin in retinal rods: the roles of rhodopsin kinase and 48-kDa protein (arrestin)

The inactivation of excited rhodopsin in the presence of ATP, rhodopsin kinase, and/or arrestin has been studied from its effect on the two subsequent steps in the light-induced enzymatic cascade: metarhodopsin II catalyzed activation of G-protein and G-protein-dependent activation of cGMP phosphodiesterase. The inactivation of G-protein (from light-scattering measurements) and that of phosphodiesterase (from measurements of cGMP hydrolysis) have been studied and compared in reconstituted systems containing various combinations of the proteins involved (rhodopsin, G-protein, phosphodiesterase, kinase, and arrestin). Our results show that rhodopsin kinase alone can terminate the activation of G-protein and that arrestin speeds up the process at a relative concentration similar to that reported in the rod (half-maximal effect at 50 nM for 4.4 microM rhodopsin). Measurements of rhodopsin phosphorylation under identical conditions show that in the presence of arrestin total metarhodopsin II inactivation is achieved when only 0.5-1.4 phosphates are bound per bleached rhodopsin, whereas in the absence of arrestin it requires binding of 12-16 phosphates per bleached rhodopsin. Phosphodiesterase activity can similarly be turned off by kinase, and the process is similarly accelerated by arrestin.     

On a soluble system for studying light activation of rod outer segment cyclic GMP phosphodiesterase

Bovine rod outer segment (ROS) cyclic GMP phosphodiesterase (PDE) could be activated about 6-fold by light, an effect that could be simulated by isolated bleached rhodopsin. About 90% of PDE activity in ROS could be extracted with 10 mM Tris-HCl, pH 7.5, but light is ineffective in activating the soluble enzyme. However, bleached rhodopsin could activate it in the presence of a very low concentration of ATP, strongly suggesting the mediation of rhodopsin in the light activation of the enzyme in ROS. Direct evidence is presented to suggest that the phosphorylation of opsin (bleached rhodopsin) is unrelated to the activation of PDE by bleached rhodopsin and ATP. The reconstitution of the light activation of PDE in a soluble system presented here opens up a new direction to future investigations on the mechanism of light regulation of cyclic GMP levels in retina and its implication in the photoreceptor function.     

Light inhibits tyrosine kinase in rod outer segments in vitro]

It was shown that short-term (10 min) light exposure of dark-adapted retinal rod outer segments (ROS) leads to a threefold inhibition of the tyrosine kinase activity. Tyrosine kinase activity in the ROS from bleached retinas is by 30% lower than in the dark-adapted ROS. Prolonged illumination (60 min) of the dark-adapted ROS restores the tyrosine kinase activity to the level of ROS from the bleached retinas.     

Light Inhibits Tyrosine Kinase Activity in Retinal Rod Outer Segments in vitro

It was shown that short-term (10 min) light exposure of dark-adapted retinal rod outer segments (ROS) leads to a threefold inhibition of the tyrosine kinase activity. Tyrosine kinase activity in the ROS from bleached retinas is by 30% lower than in the dark-adapted ROS. Prolonged illumination (60 min) of the dark-adapted ROS restores the tyrosine kinase activity to the level of ROS from the bleached retinas.     

Rapid declines in cyclic GMP of rod outer segments of intact frog photoreceptors after illumination

Considerable disagreement has resulted from experiments designed to test whether light-induced falls in cGMP in outer segment (OS) of photoreceptors precede their light-induced electrical responses. Different studies have reported initial declines at 50 ms, at s, or not at all for physiological stimuli. Such studies have employed whole retinas, isolated rod OS, or isolated rod OS with attached inner segments and involved a variety of techniques. We developed an apparatus that illuminates intact pieces of dark-adapted frog retinas at 22 degrees C for known brief durations and then rapidly (47 ms) presses their OS surface against a copper mirror cooled by liquid helium. Freezing occurs in less than 2 ms. Cyclic GMP was then assayed in cryostat sections of the OS layer. Six illumination intensities that bleached from 90 to 9 X 10(8) rhodopsin molecules per s were delivered for durations of 0.1-2 s. Compared to dark-adapted values, progressive losses of cGMP were seen with all illumination intensities. Because a significant loss in cGMP was seen after a 100 ms exposure to our dimmest stimulus, it appears that a loss of cGMP could play a role in rod visual transduction.     

Inactivation of cGMP-dependent conductance of rod outer segment plasma membrane induced by cGMP.

Integral cGMP-dependent currents as well as activity of single cGMP-activated channels in plasma membrane of rod outer segment (ROS) were recorded using the patch-clamp method. The dependence of integral currents on cGMP concentration is shown to be bell-shaped. Decrease in cGMP-dependent conductance at high cGMP concentration results from a decrease in channel opening probability. Thus, cGMP in high concentrations inactivates cGMP-dependent conductance.     

Cyclic nucleotide dependent protein kinase and the phosphorylation of endogenous proteins of retinal rod outer segments.

Cyclic nucleotide dependent protein kinase has been extracted wiht Tris or Lubrol PX from purified rod outer segments (ROS) of bovine retina. The activity of the enzyme is unaffected by light but is stimulated by either cyclic guanosine 3',5'-monophosphate (cGMP) or cyclic adenosine 3',5'-monophosphate (cAMP). Most of the solubilized enzyme elutes from DEAE-cellulose with about 0.18 M NaCl (type II protein kinase). An endogenous 30,000 molecular weight protein of the soluble fraction of ROS as well as exogenous histone are phosphorylated by the protein kinase in a cyclic nucleotide dependent manner. The Tris-extracted enzyme can be reassociated in the presence of Mg2+ with ROS membranes that are depleted of protein kinase activity. The reassociated protein kinase is insensitive to exogenous cyclic nucleotides, and it catalyzes the phosphorylation of the membrane protein, bleached rhodopsin. While the soluble and membrane-associated protein kinases may be interchangeable, they appear to be modulated by different biological signals; soluble protein kinase activity is increased by cyclic nucleotides whereas membrane-bound activity is enhanced when rhodopsin is bleached by light.     

Surfaces of rod photoreceptor disk membranes: light-activated enzymes

The light-activated GTP-binding protein (GBP) in toad rod outer segments has been located on the cytoplasmic surface (CS) of rod disk membranes by correlating biochemical results with images of quick- frozen, freeze-fractured, and deep-etched rod outer segments. This has been accomplished by selectively removing and replacing the 8-12-nm particles that are found on the CS of disk membranes, exactly in parallel with the GBP. In contrast, the large particles are not correlated with another major disk enzyme, the light-activated cGMP phosphodiesterase. We have been unable to visualize this protein. The surface density of large particles, one particle per eleven rhodopsins in isolated rod outer segments and one particle per nine rhodopsins in intact retina, correlates well with previous biochemical estimates of GBP numbers based on enzyme activity. After the identification of the large particles, we tested the effects of light on the density of particles on the surface of disk membranes in intact retinas. Retinas quick-frozen at various intervals after a bright flash of light show a modest increase (approximately 30%) in particle density by 10 s after the flash but no increase before 1 s. The number of particles on the disk membrane returns to dark levels between 1 and 10 min after the flash. The 1-s latency in the change of particle binding would appear to rule out this process as a mechanism for initiating phototransduction in the rod.