Cyclic GMP diffusion coefficient in rod photoreceptor outer segments.

Cyclic GMP (cGMP) is the intracellular messenger that mediates phototransduction in retinal rods. As photoisomerizations of rhodopsin molecules are local events, the longitudinal diffusion of cGMP in the rod outer segment should be a contributing factor to the response of the cell to light. We have employed the truncated rod outer segment preparation from bullfrog (Rana catesbeiana) and tiger salamander (Ambystoma tigrinum) to measure the cGMP diffusion coefficient. In this preparation, the distal portion of a rod outer segment was drawn into a suction pipette for measuring membrane current, and the rest of the cell was then sheared off with a glass probe, allowing bath cGMP to diffuse into the outer segment and activate the cGMP-gated channels on the surface membrane. Addition and removal of bath cGMP were fast enough to produce effectively step changes in cGMP concentration at the open end of the outer segment. When cGMP hydrolysis is inhibited by isobutylmethylxanthine (IBMX), the equation for the diffusion of cGMP inside the truncated rod outer segment has a simple analytical solution, which we have used to analyze the rise and decay kinetics of the cGMP-elicited currents. From these measurements we have obtained a cGMP diffusion coefficient of approximately 70 x 10(-8) cm2 s-1 for bullfrog rods and approximately 60 x 10(-8) cm2 s-1 for tiger salamander rods. These values are six to seven times lower than the expected value in aqueous solution. The estimated diffusion coefficient is the same at high (20-1000 microM) and low (5-10 microM) concentrations of cGMP, suggesting no significant effect from buffering over these concentration ranges.     

Dynamics of cyclic GMP synthesis in retinal rods

In retinal rods, Ca(2+) exerts negative feedback control on cGMP synthesis by guanylate cyclase (GC). This feedback loop was disrupted in mouse rods lacking guanylate cyclase activating proteins GCAP1 and GCAP2 (GCAPs(-/-)). Comparison of the behavior of wild-type and GCAPs(-/-) rods allowed us to investigate the role of the feedback loop in normal rod function. We have found that regulation of GC is apparently the only Ca(2+) feedback loop operating during the single photon response. Analysis of the rods' light responses and cellular dark noise suggests that GC normally responds to light-driven changes in [Ca(2+)] rapidly and highly cooperatively. Rapid feedback to GC speeds the rod's temporal responsiveness and improves its signal-to-noise ratio by minimizing fluctuations in cGMP.     

New insights into the fatty acid-binding protein (FABP) family in the small intestine

Cyclic GMP is essential for the ability of rods and cones to respond to the light stimuli. Light triggers hydrolysis of cGMP and stops the influx of sodium and calcium through the cGMP-gated ion channels. The consequence of this event is 2-fold: first, the decrease in the inward sodium current plays the major role in an abrupt hyperpolarization of the cellular membrane; secondly, the decrease in the Ca²⁺ influx diminishes the free intracellular Ca²⁺ concentration. While the former constitutes the essence of the phototransduction pathway in rods and cones, the latter gives rise to a potent feedback mechanism that accelerates photoreceptor recovery and adaptation to background light. One of the most important events by which Ca²⁺ feedback controls recovery and light adaptation is synthesis of cGMP by guanylyl cyclase. Two isozymes of membrane photoreceptor guanylyl cyclase (retGC) have been identified in rods and cones that are regulated by Ca²⁺-binding proteins, GCAPs. At low intracellular concentrations of Ca²⁺ typical for light-adapted rods and cones GCAPs activate RetGC, but concentrations above 500 nM typical for dark-adapted photoreceptors turn them into inhibitors of retGC. A variety of mutations found in GCAP and retGC genes have been linked to several forms of human congenital retinal diseases, such as dominant cone degeneration, cone-rod dystrophy and Leber congenital amaurosis.     

Diffusion coefficient of the cyclic GMP analog 8-(fluoresceinyl)thioguanosine 3'',5'' cyclic monophosphate in the salamander rod outer segment.

Cyclic GMP (cGMP) is the intracellular messenger mediating phototransduction in retinal rods, with its longitudinal diffusion in the rod outer segment (ROS) likely to be a factor in determining light sensitivity. From the kinetics of cGMP-activated currents in the truncated ROS of the salamander (Ambystoma tigrinum), the cGMP diffusion coefficient was previously estimated to be approximately 60 x 10(-8) cm2 s-1. On the other hand, fluorescence measurements in intact salamander ROS using 8-(fluoresceinyl)thioguanosine 3',5'-cyclic monophosphate (Fl-cGMP) led to a diffusion coefficient for this compound of 1 x 10(-8) cm2 s-1; after corrections for differences in size and in binding to cellular components between cGMP and Fl-cGMP, this gave an upper limit of 11 x 10(-8) cm2 s-1 for the cGMP diffusion coefficient. To properly compare the two sets of measurements, we have examined the diffusion of Fl-cGMP in the truncated ROS. From the kinetics of Fl-cGMP-activated currents, we have obtained a diffusion coefficient of 3 x 10(-8) cm2 s-1 for this analog; the cGMP diffusion coefficient measured from the same truncated ROSs was approximately 80 x 10(-8) cm2 s-1. Thus, a factor of 27 appears appropriate for correcting differences in size and intracellular binding between cGMP and Fl-cGMP. Application of this correction factor to the Fl-cGMP diffusion coefficient measurements by Olson and Pugh (1993) gives a cGMP diffusion coefficient of approximately 30 x 10(-8) cm2 s-1, in reasonable agreement with the value measured from the truncated ROS.     

Developmental regulation of calcium-dependent feedback in Xenopus rods

The kinetics of activation and inactivation in the phototransduction pathway of developing Xenopus rods were studied. The gain of the activation steps in transduction (amplification) increased and photoresponses became more rapid as the rods matured from the larval to the adult stage. The time to peak was significantly shorter in adults (1.3 s) than tadpoles (2 s). Moreover, adult rods recovered twice as fast from saturating flashes than did larval rods without changes of the dominant time constant (2.5 s). Guanylate cyclase (GC) activity, determined using IBMX steps, increased in adult rods from approximately 1.1 s(-1) to 3.7 s(-1) 5 s after a saturating flash delivering 6,000 photoisomerizations. In larval rods, it increased from 1.8 s(-1) to 4.0 s(-1) 9 s after an equivalent flash. However, the ratio of amplification to the measured dark phosphodiesterase activity was constant. Guanylate cyclase-activating protein (GCAP1) levels and normalized Na+/Ca2+, K+ exchanger currents were increased in adults compared with tadpoles. Together, these results are consistent with the acceleration of the recovery phase in adult rods via developmental regulation of calcium homeostasis. Despite these large changes, the single photon response amplitude was approximately 0.6 pA throughout development. Reduction of calcium feedback with BAPTA increased adult single photon response amplitudes threefold and reduced its cutoff frequency to that observed with tadpole rods. Linear mathematical modeling suggests that calcium-dependent feedback can account for the observed differences in the power spectra of larval and adult rods. We conclude that larval Xenopus maximize sensitivity at the expense of slower response kinetics while adults maximize response kinetics at the expense of sensitivity.     

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.     

A role for GCAP2 in regulating the photoresponse. Guanylyl cyclase activation and rod electrophysiology in GUCA1B knock-out mice

Cyclic GMP serves as the second messenger in visual transduction, linking photon absorption by rhodopsin to the activity of ion channels. Synthesis of cGMP in photoreceptors is supported by a pair of retina-specific guanylyl cyclases, retGC1 and -2. Two neuronal calcium sensors, GCAP1 and GCAP2, confer Ca(2+) sensitivity to guanylyl cyclase activity, but the importance and the contribution of each GCAP is controversial. To explore this issue, the gene GUCA1B, coding for GCAP2, was disrupted in mice, and the capacity for knock-out rods to regulate retGC and generate photoresponses was tested. The knock-out did not compromise rod viability or alter outer segment ultrastructure. Levels of retGC1, retGC2, and GCAP-1 expression did not undergo compensatory changes, but the absence of GCAP2 affected guanylyl cyclase activity in two ways; (a) the maximal rate of cGMP synthesis at low [Ca(2+)] dropped 2-fold and (b) the half-maximal rate of cGMP synthesis was attained at a higher than normal [Ca(2+)]. The addition of an antibody raised against mouse GCAP2 produced similar effects on the guanylyl cyclase activity in wild type retinas. Flash responses of GCAP2 knock-out rods recovered more slowly than normal. Knock-out rods became more sensitive to flashes and to steps of illumination but tended to saturate at lower intensities, as compared with wild type rods. Therefore, GCAP2 regulation of guanylyl cyclase activity quickens the recovery of flash and step responses and adjusts the operating range of rods to higher intensities of ambient illumination.     

Photoreceptors of the retina and pinealocytes of the pineal gland share common components of signal transduction

Light absorbed by retinal photoreceptors triggers a cascade of reactions that initiate cGMP hydrolysis, cation channel closure and membrane hyperpolarization. Down-regulation of the cascade involves additional proteins that interfere with amplification along the cascade. Pinealocytes are activated by norepinephrine during the dark phase of the day/night cycle. Mature pinealocytes of the mammalian pineal express the known photoreceptor proteins that are implicated in down-regulation of the visual cascade, but the cascade components that produce cGMP hydrolysis and membrane hyperpolarization are absent. Pinealocytes accumulate cyclic AMP minimally when norepinephrine activates their beta adrenergic receptors alone, but the response is potentiated by the simultaneous activation of their alpha-1 adrenergic receptors. A model is proposed whereby phosducin, a phosphoprotein that binds the beta, gamma subunit of G-proteins, could modulate the synthesis of cyclic AMP by buffering the amount of beta, gamma G-protein subunits that are available for activating adenylate cyclase.Special issue dedicated to Dr. Frederick E. Samson.     

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.     

Diffusion coefficient of cyclic GMP in salamander rod outer segments estimated with two fluorescent probes.

Experiments have demonstrated that single photoisomerizations in amphibian and primate rods can cause the suppression of 3-5% of the dark circulating current at the response peak (Baylor, D. A., T. D. Lamb, and K. W. Yau. 1979. J. Physiol. (Lond.). 288:613-634; Baylor, D. A., B. J. Nunn, and J. L. Schnapf. 1984. J. Physiol. (Lond.). 357:575-607). These results indicate that the change in [cGMP] effected by a single isomerization must spread longitudinally over at least the corresponding fractional length of the outer segment. The effective longitudinal diffusion coefficient, Dx, of cGMP is thus an important determinant of rod sensitivity. We report here measurements of the effective longitudinal diffusion coefficients, Dx, of two fluorescently labeled molecules: 5/6-carboxyfluorescein and 8-(fluoresceinyl)thioguanosine 3',5'-cyclic monophosphate, introduced into detached outer segments via whole-cell patch electrodes. For these compounds, the average time for equilibration of the entire outer segment with the patch pipette was approximately 6 min. Fluorescence images of rods were analyzed with a one-dimensional diffusion model that included limitations on transfer between the electrode and outer segment and the effects of intracellular binding of the dyes. The analyses yielded estimates of Dx of 1.9 and 1.0 microns 2.s-1 for the two dyes. It is shown that these results place an upper limit on Dx for cGMP of 11 microns2.s-1. The actual value of Dx for cGMP in the rod will depend on the degree of intracellular binding of cGMP. Estimates of the effective buffering power for cGMP in the rod at rest range from two to six (Lamb and Pugh, 1992; Cote and Brunnock, 1993). When combined with these estimates, our results predict that for cGMP itself, Dx falls within the range of 1.4-5.5 microns 2.s-1.     

Bicarbonate Modulates Photoreceptor Guanylate Cyclase (ROS-GC) Catalytic Activity

By generating the second messenger cGMP in retinal rods and cones, ROS-GC plays a central role in visual transduction. Guanylate cyclase-activating proteins (GCAPs) link cGMP synthesis to the light-induced fall in [Ca²⁺]_i to help set absolute sensitivity and assure prompt recovery of the response to light. The present report discloses a surprising feature of this system: ROS-GC is a sensor of bicarbonate. Recombinant ROS-GCs synthesized cGMP from GTP at faster rates in the presence of bicarbonate with an ED₅₀ of 27 mm for ROS-GC1 and 39 mm for ROS-GC2. The effect required neither Ca²⁺ nor use of the GCAPs domains; however, stimulation of ROS-GC1 was more powerful in the presence of GCAP1 or GCAP2 at low [Ca²⁺]. When applied to retinal photoreceptors, bicarbonate enhanced the circulating current, decreased sensitivity to flashes, and accelerated flash response kinetics. Bicarbonate was effective when applied either to the outer or inner segment of red-sensitive cones. In contrast, bicarbonate exerted an effect when applied to the inner segment of rods but had little efficacy when applied to the outer segment. The findings define a new regulatory mechanism of the ROS-GC system that affects visual transduction and is likely to affect the course of retinal diseases caused by cGMP toxicity.     

Rod outer segment structure influences the apparent kinetic parameters of cyclic GMP phosphodiesterase

Cyclic GMP hydrolysis by the phosphodiesterase (PDE) of retinal rod outer segments (ROS) is a key amplification step in phototransduction. Definitive estimates of the turnover number, kcat, and of the Km are crucial to quantifying the amplification contributed by the PDE. Published estimates for these kinetic parameters vary widely; moreover, light-dependent changes in the Km of PDE have been reported. The experiments and analyses reported here account for most observed variations in apparent Km, and they lead to definitive estimates of the intrinsic kinetic parameters in amphibian rods. We first obtained a new and highly accurate estimate of the ratio of holo-PDE to rhodopsin in the amphibian ROS, 1:270. We then estimated the apparent kinetic parameters of light-activated PDE of suspensions of disrupted frog ROS whose structural integrity was systematically varied. In the most severely disrupted ROS preparation, we found Km = 95 microM and kcat = 4,400 cGMP.s-1. In suspensions of disc-stack fragments of greater integrity, the apparent Km increased to approximately 600 microM, though kcat remained unchanged. In contrast, the Km for cAMP was not shifted in the disc stack preparations. A theoretical analysis shows that the elevated apparent Km of suspensions of disc stacks can be explained as a consequence of diffusion with hydrolysis in the disc stack, which causes active PDEs nearer the center of the stack to be exposed to a lower concentration of cyclic GMP than PDEs at the disc stack rim. The analysis predicts our observation that the apparent Km for cGMP is elevated with no accompanying decrease in kcat. The analysis also predicts the lack of a Km shift for cAMP and the previously reported light dependence of the apparent Km for cGMP. We conclude that the intrinsic kinetic parameters of the PDE do not vary with light or structural integrity, and are those of the most severely disrupted disc stacks.     

Regulation of Mammalian Cone Phototransduction by Recoverin and Rhodopsin Kinase

Cone photoreceptors function under daylight conditions and are essential for color perception and vision with high temporal and spatial resolution. A remarkable feature of cones is that, unlike rods, they remain responsive in bright light. In rods, light triggers a decline in intracellular calcium, which exerts a well studied negative feedback on phototransduction that includes calcium-dependent inhibition of rhodopsin kinase (GRK1) by recoverin. Rods and cones share the same isoforms of recoverin and GRK1, and photoactivation also triggers a calcium decline in cones. However, the molecular mechanisms by which calcium exerts negative feedback on cone phototransduction through recoverin and GRK1 are not well understood. Here, we examined this question using mice expressing various levels of GRK1 or lacking recoverin. We show that although GRK1 is required for the timely inactivation of mouse cone photoresponse, gradually increasing its expression progressively delays the cone response recovery. This surprising result is in contrast with the known effect of increasing GRK1 expression in rods. Notably, the kinetics of cone responses converge and become independent of GRK1 levels for flashes activating more than ∼1% of cone pigment. Thus, mouse cone response recovery in bright light is independent of pigment phosphorylation and likely reflects the spontaneous decay of photoactivated visual pigment. We also find that recoverin potentiates the sensitivity of cones in dim light conditions but does not contribute to their capacity to function in bright light.     

The small GTPase HRas shapes local PI3K signals through positive feedback and regulates persistent membrane extension in migrating fibroblasts

Self-amplification of phosphoinositide 3-kinase (PI3K) signaling is believed to regulate asymmetric membrane extension and cell migration, but the molecular organization of the underlying feedback circuit is elusive. Here we use an inducible approach to synthetically activate PI3K and interrogate the feedback circuitry governing self-enhancement of 3′-phosphoinositide (3-PI) signals in NIH3T3 fibroblasts. Synthetic activation of PI3K initially leads to uniform production of 3-PIs at the plasma membrane, followed by the appearance of asymmetric and highly amplified 3-PI signals. A detailed spatiotemporal analysis shows that local self-amplifying 3-PI signals drive rapid membrane extension with remarkable directional persistence and initiate a robust migratory response. This positive feedback loop is critically dependent on the small GTPase HRas. Silencing of HRas abrogates local amplification of 3-PI signals upon synthetic PI3K activation and results in short-lived protrusion events that do not support cell migration. Finally, our data indicate that this feedback circuit is likely to operate during platelet-derived growth factor–induced random cell migration. We conclude that positive feedback between PI3K and HRas is essential for fibroblasts to spontaneously self-organize and generate a productive migratory response in the absence of spatial cues.     

Calcium feedback and sensitivity regulation in primate rods.

Membrane current was recorded from a single primate rod with a suction pipette while the cell was bath perfused with solutions maintained at a temperature of approximately 38 degrees C. A transient inward current was observed at the onset of bright illumination after briefly exposing the outer segment in darkness to Ringer's (Locke) solution containing 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of cGMP phosphodiesterase. After briefly removing external Na+ from around the outer segment in darkness, a similar current was observed upon Na+ restoration in bright light. By analogy to amphibian rods, this inward current was interpreted to represent the activity of an electrogenic Na(+)-dependent Ca2+ efflux, which under physiological conditions in the light is expected to reduce the free Ca2+ in the outer segment and provide negative feedback (the "Ca2+ feedback") to the phototransduction process. The exchange current had a saturated amplitude of up to approximately 5 pA and a decline time course that appeared to have more than one exponential component. In the absence of the Ca2+ feedback, made possible by removing the Ca2+ influx and efflux at the outer segment using a 0 Na(+)-0 Ca2+ external solution, the response of a rod to a dim flash was two to three times larger and had a longer time to peak than in physiological solution. These changes can be approximately accounted for by a simple model describing the Ca2+ feedback in primate rods. The dark hydrolytic rate for cGMP was estimated to be 1.2 s-1. The incremental hydrolytic rate, beta*(t), activated by one photoisomerization was approximately 0.09 s-1 at its peak, with a time-integrated activity, integral of beta*(t)dt, of approximately 0.033, both numbers being derived assuming spatial homogeneity in the outer segment. Finally, we have found that primate rods adapt to light in much the same way as amphibian and other mammalian rods, such as showing a Weber-Fechner relation between flash sensitivity and background light. The Ca2+ feedback model we have constructed can also explain this feature reasonably well.     

The cGMP/protein kinase G pathway contributes to dihydropyridine-sensitive calcium response and cytokine production in TH2 lymphocytes

Th2 lymphocytes differ from other CD4+ T lymphocytes not only by their effector tasks but also by their T cell receptor (TCR)-dependent signaling pathways. We previously showed that dihydropyridine receptors (DHPR) involved in TCR-induced calcium inflow were selectively expressed in Th2 cells. In this report, we studied whether cGMP-dependent protein kinase G (PKG) activation was implicated in the regulation of DHPR-dependent calcium response and cytokine production in Th2 lymphocytes. The contribution of cGMP in Th2 signaling was supported by the following results: 1) TCR activation elicited cGMP production, which triggered calcium increase responsible for nuclear factor of activated T cell translocation and Il4 gene expression; 2) guanylate cyclase activation by nitric oxide donors increased intracellular cGMP concentration and induced calcium inflow and IL-4 production; 3) reciprocally, guanylate cyclase inhibition reduced calcium response and Th2 cytokine production associated with TCR activation. In addition, DHPR blockade abolished cGMP-induced [Ca2+]i increase, indicating that TCR-induced DHP-sensitive calcium inflow is dependent on cGMP in Th2 cells. Th2 lymphocytes from PKG1-deficient mice displayed impaired calcium signaling and IL-4 production, as did wild-type Th2 cells treated with PKG inhibitors. Altogether, our data indicate that, in Th2 cells, cGMP is produced upon TCR engagement and activates PKG, which controls DHP-sensitive calcium inflow and Th2 cytokine production.     

Calcium-independent and cAMP-dependent modulation of soluble guanylyl cyclase activity by G protein-coupled receptors in pituitary cells

It is well established that G protein-coupled receptors stimulate nitric oxide-sensitive soluble guanylyl cyclase by increasing intracellular Ca(2+) and activating Ca(2+)-dependent nitric-oxide synthases. In pituitary cells receptors that stimulated adenylyl cyclase, growth hormone-releasing hormone, corticotropin-releasing factor, and thyrotropin-releasing hormone also stimulated calcium signaling and increased cGMP levels, whereas receptors that inhibited adenylyl cyclase, endothelin-A, and dopamine-2 also inhibited spontaneous calcium transients and decreased cGMP levels. However, receptor-controlled up- and down-regulation of cyclic nucleotide accumulation was not blocked by abolition of Ca(2+) signaling, suggesting that cAMP production affects cGMP accumulation. Agonist-induced cGMP accumulation was observed in cells incubated in the presence of various phosphodiesterase and soluble guanylyl cyclase inhibitors, confirming that G(s)-coupled receptors stimulated de novo cGMP production. Furthermore, cholera toxin (an activator of G(s)), forskolin (an activator of adenylyl cyclase), and 8-Br-cAMP (a permeable cAMP analog) mimicked the stimulatory action of G(s)-coupled receptors on cGMP production. Basal, agonist-, cholera toxin-, and forskolin-stimulated cGMP production, but not cAMP production, was significantly reduced in cells treated with H89, a protein kinase A inhibitor. These results indicate that coupling seven plasma membrane-domain receptors to an adenylyl cyclase signaling pathway provides an additional calcium-independent and cAMP-dependent mechanism for modulating soluble guanylyl cyclase activity in pituitary cells.     

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.     

An intracellular modulator of the 3'5'-cGMP hydrolysis in growing Dictyostelium discoideum amoebae

A heat-stable and acid-stable macromolecular factor present in the cytosol of growing Dictyostelium discoideum amoebae affects specifically the intracellular cGMP phosphodiesterase. It decreases the V of the enzyme but does not alter its Km. It has no effect on the cAMP or cGMP hydrolysis catalyzed by the intracellular cAMP-cGMP phosphodiesterases or by the extracellular phosphodiesterase. It is also expressed in a mutant (HPX235), defective in the synthesis of the cAMP-cGMP phosphodiesterases but capable of intracellular transduction of the chemotactic signal. This factor is resistant to several nucleases, proteases and phospholipases, and has an apparent molecular weight between 3500-10000. In contrast, the protein phosphodiesterase inhibitor secreted by the amoebae exerts an opposite inhibition on the intracellular phosphodiesterases. These two inhibitory factors may regulate intracellular cGMP hydrolysis during the chemotactic response.     

Mathematical Analysis of Spontaneous Emergence of Cell Polarity

Cell polarization, in which intracellular substances are asymmetrically distributed, enables cells to carry out specialized functions. While cell polarity is often induced by intracellular or extracellular spatial cues, spontaneous polarization (the so-called symmetry breaking) may also occur in the absence of spatial cues. Many computational models have been used to investigate the mechanisms of symmetry breaking, and it was proved that spontaneous polarization occurs when the lateral diffusion of inactive signaling molecules is much faster than that of active signaling molecules. This conclusion leaves an important question of how, as observed in many biological systems, cell polarity emerges when active and inactive membrane-bound molecules diffuse at similar rates while cycling between cytoplasm and membrane takes place. The recent studies of Rätz and Röger showed that, when the cytosolic and membrane diffusion are very different, spontaneous polarization is possible even if the membrane-bound species diffuse at the same rate. In this paper, we formulate a two-equation non-local reaction-diffusion model with general forms of positive feedback. We apply Turing stability analysis to identify parameter conditions for achieving cell polarization. Our results show that spontaneous polarization can be achieved within some parameter ranges even when active and inactive signaling molecules diffuse at similar rates. In addition, different forms of positive feedback are explored to show that a non-local molecule-mediated feedback is important for sharping the localization as well as giving rise to fast dynamics to achieve robust polarization.