Calcium modulation of cyclic GMP synthesis in rat visual cells

The synthesis of cyclic GMP in dark-adapted rat retinas, retinal homogenates or isolated ROS is stimulated during incubation with medium containing low levels of Ca2+. The guanylate cyclase that is stimulated by low [Ca2+] is localized exclusively in visual cells of the retina because the stimulatory effect of low [Ca2+] is observed in developing retinas only after visual cells begin to differentiate, and it is lost in diseased retinas when the photoreceptor cells degenerate. The accumulation of cyclic GMP during incubation with low [Ca/+] is prevented by illumination; the effect of light stems apparently from the light-enhanced hydrolysis of cyclic GMP. Following light adaptation and transfer of the animals to darkness, retinas become progressively more responsive to low [Ca2+], and a maximal response is restored after about 30 min of dark adaptation in vivo. Incubated retinas accumulate cyclic GMP when exposed to media containing less than about 5 x 10(-9) M [Ca2+], whereas the synthesis of cyclic GMP in retinal homogenates or lysed ROS is stimulated at concentrations of less than 10(-6) M-Ca2+. These findings indicate that calcium acts as an inhibitory effector in the regulation of guanylate cyclase in rod photoreceptor cells, and they suggest that changes in intracellular [Ca2+] may regulate the synthesis of cyclic GMP in dark-adapted visual cells in situ.     

Response of rabbit ciliary process cyclic nucleotides to specific phosphodiesterase inhibitors

PURPOSE: To determine which cyclic nucleotide phosphodiesterase family activities can be identified in rabbit ciliary processes. METHODS: Freshly excised rabbit ciliary processes were incubated in vitro with family selective phosphodiesterase inhibitors in the absence or presence of activators of soluble or membrane bound guanylate cyclase. The resulting increases of cyclic AMP and cyclic GMP were measured by RIA. RESULTS: Rabbit ciliary process cyclic AMP levels were increased by the phosphodiesterase 1 and 4 selective inhibitors, 8-methoxymethyl-IBMX and rolipram, respectively. Cyclic GMP levels were increased by 8-methoxymethyl-IBMX; whereas the phosphodiesterase 5 and 6 selective inhibitor, zaprinast, increased cyclic GMP little. Nitric oxide donors increased cyclic AMP in addition to cyclic GMP, and inhibition of soluble guanylate cyclase eliminated the increase of cyclic AMP. The effects of sodium nitroprusside and the phosphodiesterase 3 selective inhibitor, cilostamide, on cyclic AMP were not additive suggesting that the sodium nitroprusside mediated increase of cyclic GMP, like cilostamide, inhibited phosphodiesterase 3. CONCLUSIONS: Cyclic AMP in rabbit ciliary processes is hydrolyzed primarily by phosphodiesterases 1 and 4, and, when cyclic GMP levels are low, by phosphodiesterase 3; cyclic GMP is hydrolyzed primarily by phosphodiesterase 1.     

Inherited rod-cone dysplasia: abnormal distribution of cyclic GMP in visual cells of affected Irish setters

Light-adapted retinas from Irish setters affected with inherited rod-cone dysplasia accumulate high concentrations of cyclic GMP in the outer plexiform layer (OPL). A 29-fold difference in OPL cGMP levels between affected and normal occurs at 19-20 days. However, the highest concentration, 550 mumol kg-1 (dry) wt, is reached at about 4 weeks of age, at which time OPL cyclic GMP is 20-fold higher than cGMP in the OPL of normal control retinas. Levels remain high in affected OPL until about 7 weeks of age; after this, the cyclic GMP level falls and the peak shifts to the outer nuclear layer (ONL). In the normal retina on the other hand, the highest cyclic GMP levels are in the outer segments (OS). The result is that cyclic GMP is highest at opposite ends of the photoreceptor neuron in normal and affected retinas during the first 7 weeks of development. These differing distributions are established as early as postnatal day 10, before signs of degeneration become evident, as determined by either ERG or morphological examination. Moreover, a 38-fold rise in cyclic GMP occurs in the ONL of affected retinas between 1 and 3 weeks of age significantly before the degeneration of nuclei begins at 4 to 5 weeks. The early differences in cyclic GMP distribution are probably not due to differences in guanylate cyclase activity since enzyme levels in normal and affected photoreceptor cells are virtually identical until about 4 weeks of age. Since cGMP has been observed to reach high levels in normal dark-adapted rabbit and frog OPL, the extraordinary OPL levels seen in affected setters may indicate the importance of cGMP in both normal synaptic function and in the disease process.     

Rods are selectively altered by lead: I. Electrophysiology and biochemistry

In vitro studies have demonstrated that lead selectively and reversibly depresses the rod photoreceptor component of the electroretinogram (ERG). To determine if low-level lead exposure during early postnatal development produced long-term selective rod deficits, we examined rod and cone ERG functions and cyclic GMP and cyclic AMP metabolism in adult control and lead-exposed rats. A-wave and b-wave voltage-log intensity and latency-log intensity functions, generated from single-flash ERGs in fully dark-adapted rats, revealed that low-level lead exposure during early postnatal development caused a 23- and 18% decrease in maximum amplitude, a 1.0- and 0.5 log unit decrease in absolute sensitivity and a mean latency increase of 47- and 29%, respectively. Additional ERG experiments, using scotopically balanced stimuli and scotopic and photopic flicker fusion frequency functions, also demonstrated selective rod deficits. Cone ERGs, elicited by 30-Hz white flashes in the presence of a white background adapting light, were similar in control and lead-exposed rats. Lead exposure during early postnatal development caused cGMP levels in dark-adapted and light-adapted retinas to increase 40- and 25%, respectively, above controls whereas cyclic AMP levels remained unchanged. Light-activated cyclic GMP phosphodiesterase (cGMP-PDE) was inhibited 40% while guanylate cyclase activity was unchanged. The retinal lead concentration was 10(-6) M at the end of exposure (day 21) while at the time of ERG testing and biochemical analysis it was 10(-7) M. In vitro studies with adult control retinas incubated with 10(-9)-10(-4) M lead revealed a dose-response inhibition (10-40%) of cGMP-PDE between 10(-6)- and 10(-4) M lead and stimulation of guanylate cyclase (20-158%) only above 10(-4) M lead, indicating that cGMP-PDE is more sensitive to the direct effects of lead than the synthetic cGMP enzyme. These in vitro cyclic nucleotide metabolism results are similar to those we observed in vivo and both are consistent with the observed ERG changes. The selective rod-mediated amplitude, sensitivity and temporal deficits and the lack of effect on the cone ERGs clearly demonstrate that low-level lead exposure during early postnatal development causes a long-term selective disruption of rat rod photoreceptors. The relevance and applicability of these data to subclinical pediatric lead poisoning has yet to be established.     

Cyclic GMP in the retinas of normal mice and those heterozygous for early-onset photoreceptor dystrophy

Cyclic GMP metabolism has been investigated in the retinas of mice that are heterozygous for a 'photoreceptor dystrophy' gene and have a lowered concentration of cGMP in their photoreceptor cells. The concentration of rhodopsin, retinal morphology and guanylate cyclase kinetics were normal. Cyclic GMP phosphodiesterase had a lowered affinity for cGMP. In accord with previous observations, chelation of exogenous calcium had no effect on cGMP levels in light-adapted retinas but increased them in dark-adapted tissue. The difference between cGMP concentrations in heterozygous and normal retinas in the dark was then eliminated. It was concluded that a modulator of cGMP phosphodiesterase activity is most likely to be causing the lowered steady-state level of cGMP in heterozygous retinas and that calcium is not involved.     

Effects of low AIPL1 expression on phototransduction in rods

PURPOSE: To investigate the impact of aryl hydrocarbon receptor-interacting protein-like (AIPL)-1 on photoreception in rods. METHODS: Photoresponses of mouse rods expressing lowered amounts of AIPL1 were studied by single-cell and electroretinogram (ERG) recordings. Phototransduction protein levels and enzymatic activities were determined in biochemical assays. Ca2+ dynamics were probed with a fluorescent dye. Comparisons were made to rods expressing mutant Y99C guanylate cyclase activating protein (GCAP)-1, to understand which effects arose from elevated dark levels of cGMP and Ca2+. RESULTS: Except for PDE, transduction protein levels were normal in low-AIPL1 retinas, as were guanylate cyclase (GC), rhodopsin kinase (RK), and normalized phosphodiesterase (PDE) activities. Y99C and low-AIPL1 rods were more sensitive to flashes than normal, but flash responses of low-AIPL1 rods showed an abnormal delay, reduced rate of increase, and longer recovery not present in Y99C rod responses. In addition, low-AIPL1 rods but not Y99C rods failed to reach the normal light-induced minimum in Ca2+ concentration. CONCLUSIONS: Reduced AIPL1 delayed the photoresponse, decreased its amplification constant, slowed a rate-limiting step in its recovery, and limited the light-induced decrease in Ca2+. Not all changes were attributable to decreased PDE or to elevated cGMP and Ca2+ in darkness. Therefore, AIPL1 directly or indirectly affects more than one component of phototransduction.     

Increased intraocular pressure following topical azide or nitroprusside.

In rabbits the topical administration of sodium azide (NaNs) or sodium nitroprusside (SNP) increased intraocular pressure in a dose-response manner. These agents, which activate guanylate cyclase, elevated cyclic GMP in the aqueous humor. Systemic blood pressure and pulse were not altered. Tonographic outflow facility was unchanged, suggesting an increase in aqueous humor flow as the mechanism for the elevation of intraocular pressure. Posterior chamber aqueous humor ascorbate concentration was decreased in the eye receiving the NaN3 or SNP. Systemic pretreatment with phenoxybenzamine, an alpha-adrenergic blocking agent, prevented the elevation of intraocular pressure observed following NaN3 and SNP. Pretreatment with systemic indomethacin, propranolol, or acetazolamide or the topical application of atropine or epinephrine failed to alter the elevation of intraocular pressure by either NaN3 or SNP.     

Nitric oxide/guanylate cyclase pathways and flow in anterior segment perfusion

BACKGROUND: The nitric oxide/guanylate cyclase pathway has been suggested to participate in the regulation of intraocular pressure. In the present study, the involvement of nitric oxide pathways on the outflow through the trabecular meshwork was assessed using pharmacological manipulation of the nitric oxide pathway. METHODS: Anterior segments of human donor eyes were maintained in an organ culture perfusion system, and the effects of L-NAME, an inhibitor of nitric oxide synthase, on the flow rate was determined. In a second series, the effects of consecutive application of L-arginine as substrate for nitric oxide synthase, L-NAME, and sodium nitroprusside, a nitric oxide-donor, were studied. The cyclic GMP levels in the perfusate were assessed with an ELISA immunoassay kit. RESULTS: In the first series of experiments, L-NAME caused a statistically significant decrease in flow rate of 10%, accompanied by a decrease in cGMP levels. In the second series, L-arginine did not alter flow, and the effect of L-NAME seen in the first series was prevented by the high preload of L-arginine. Nitroprusside caused a significant 10% increase of flow rate. In the perfusate, cGMP levels were not altered by L-arginine and L-NAME, but were increased after nitroprusside. CONCLUSION: Under organ culture perfusion conditions, modulation of the nitric oxide/guanylate cyclase system alters the flow rate through the trabecular meshwork within a total range of 20%; i.e. the difference between inhibition of NO synthesis and the presence of a NO-donor. These results indicate that the nitric oxide/guanylate cyclase system plays a role in aqueous humour dynamics and, therefore, in the regulation of intraocular pressure.     

Inhibitors of guanylate cyclase inhibit phototransduction in limulus ventral photoreceptors

The second messenger systems involved in the final stages of the phototransduction cascade in Limulus photoreceptors remain unclear. Excised patches of transducing membrane contain cGMP-gated channels, suggesting the involvement of cGMP in the excitation process. To further explore this possibility, we tested the effects of inhibitors and agonists of guanylate cyclase. The active site cyclase inhibitors guanosine 5'-tetraphosphate and adenosine 5'-tetraphosphate produced a reversible reduction of the response to light without affecting resting membrane properties. The cyclase inhibitor Rp-GTPalphaS produced a similar reduction, but the effect was only slightly reversible. The reduction in the response produced by these inhibitors was robust, often producing over a 95% decrease in the amplitude of the light response. Previous work had shown that an end-product cyclase inhibitor, imidodiphosphate, also inhibited the response. The consistent results with four different guanylate cyclase inhibitors strongly support the involvement of this enzyme in the phototransduction cascade. To determine whether the guanylate cyclase involved is the NO-dependent soluble form, we applied inhibitors and activators of the nitric oxide synthase/guanylate cyclase pathway such as L-N5-(1-iminoethyl) ornithine, sodium nitroprusside, and carboxy-PTIO. None of these agents had any substantial effect on phototransduction. Taken together, these results support a role for a particulate guanylate cyclase in Limulus photoreceptor excitation.     

Nitric oxide and cyclic GMP-mediated protein secretion from cultured lacrimal gland acinar cells.

PURPOSE: Nitric oxide (NO) donors and NO synthase (NOS) substrates were tested for their use to stimulate protein secretion from cultured lacrimal gland acinar cells, through activation of guanylate cyclase. METHOD: Rabbit lacrimal gland epithelial cells (RLG cells) were incubated with NO donors and/or NOS substrates and the protein released into culture medium was determined with bicinchoninic acid assay. Guanylate cyclase activation by NO precursors was determined by measurement of c-GMP produced. RESULTS: Both NO donors and NOS substrates were able to stimulate protein release from RLG cells. Among 6 compounds studied, sodium nitroprusside, isosorbide dinitrate and N(a)-benzoyl L-arginine ethyl ester (BAEE) were most potent to release protein over 100% of the basal release. The guanylate cyclase activity was stimulated by these NO precursors and was inhibited by guanylate cyclase inhibitor, [1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ). CONCLUSION: NO donors and NOS substrates were able to stimulate protein release from RLG cells via activation of guanylate cyclase and c-GMP release, which was blocked by guanylate cyclase inhibitor, ODQ. It indicates that NO donors and NOS substrates could be used for the treatment of dry eye syndrome if the same holds true in dry eye animal models.     

Correlations between neuronal nitric oxide synthase and muscarinic M3/M1 receptors in the rat retina

This study determined the different signal pathways involved in M1/M3 muscarinic acetylcholine receptor (mAChR) dependent stimulation of nitric oxide synthase (NOS) activity/cyclic GMP (cGMP) production and nNOS mRNA expression in rat retina. Exposure of the retina to different concentrations of carbachol caused an increase in NOS activity, cGMP production and phosphoinositol (PI) accumulation. The increase in NOS activity and cGMP content was blocked by L-NMMA and ODQ, respectively. Also, phospholipase C (PLC) and calcium/calmodulin (CaM) inhibition prevented the carbachol activation on NOS/cGMP pathways. Both, 4-DAMP and pirenzepine but not AF-DX 116 blocked the increase in NOS and cGMP induced by carbachol. Carbachol-stimulation of M1/M3 mAChR increased nNOS-mRNA levels associated with an increase of endogenous NO and cGMP production. The mechanism appears to occur secondarily to stimulation of PIs turnover via PLC. This triggers a cascade reaction involving CaM and soluble guanylate cyclase leading to NO and cGMP accumulation, that in turn, up regulates nNOS-mRNA gene expression. These results give novel insight into the mechanism involved in the regulation of nNOS-mRNA levels by mAChR activation of retina.     

Regulation of lens cyclic nucleotide metabolism by Ca2+ plus calmodulin

Adenylate cyclase activity was identified in membranes isolated from bovine lens fiber cells. Basal activity, in the presence of microM Ca2+ was stimulated by either sodium fluoride, guanosine 5'-[alpha,beta-imido]triphosphate (Gpp(NH)p), or forskolin; ethylene glycolbis(2-aminoethylether) tetraacetic acid (EGTA) markedly inhibited both the basal activity and the extent of stimulation by these agents. Exogenous calmodulin enhanced the Ca2+-dependent stimulation of adenylate cyclase activity. In the presence of optimal concentrations of Ca2+ plus calmodulin, adenylate cyclase activity was approximately 15 times greater than that in the presence of EGTA. Adenylate cyclase activity was not stimulated by a number of potential agonists that included carbachol, serotonin, prostaglandin E1 (PGE1), prostaglandin E2 (PGE2), adenosine, isoproterenol epinephrine, dopamine, and phenylephrine. The presence of the Ns and Ni guanine nucleotide regulatory complexes was indicated by two observations: Cholera toxin catalyzed the adenosine diphosphate (ADP) ribosylation of a number of lens membrane proteins, including a 46,500-dalton component (likely the alpha-subunit of Ns), and Pertussis toxin catalyzed the ADP ribosylation of a single 41,000-dalton lens membrane component (likely the alpha-subunit of Ni). However, that Gpp(NH)p did not inhibit either the forskolin-activated or the calmodulin-activated adenylate cyclase activities does not indicate a role for Ni in regulating this enzyme. Both cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) phosphodiesterase activities were identified in a supernate fraction derived from bovine lens. The cAMP phosphodiesterase activity appeared to be predominantly the low Km form of the enzyme. The cGMP phosphodiesterase activity, which was Ca2+-dependent, was partly inhibited maximally by 7 microM R24571, indicating its probable calmodulin dependence.(ABSTRACT TRUNCATED AT 250 WORDS)     

Degradation and resynthesis of cyclic 3', 5' -guanosine monophosphate in truncated rod photoreceptors from bovine retina

Truncated rod photoreceptors containing outer segments with inner segment attachments were prepared from bovine retinas. In the absence of a high-energy donor, the preparation transformed cyclic GMP to 5′-GMP, guanosine and a small amount of cyclic XMP. In the presence of ATP. the preparation formed GDP and GTP from 5′-GMP, and GTP endogenously formed was converted to cyclic GMP. Conditions for optimal activity of the above reactions were defined and a rank ordering of the specific activities of the reactions established. The specific activity of each enzyme was greater in truncated photoreceptors than in homogenates of retina. The efficiency of the metabolic cycle to resynthesize cyclic GMP from 5′-GMP in vivo probably depends upon the energy state of the visual cell and upon the degree of compartmentation of substrates within the inner and outer segment of the photoreceptor.     

Purification and characterization of calmodulin-dependent functional protein, phosphodiesterase, in the lens

Calmodulin and calmodulin-dependent functional protein play an important role in the maintenance of lens transparency and homeostasis. In the present study, phosphodiesterase, one of the typical calmodulin-dependent functional proteins, was purified from bovine lens by DEAE-cellulose chromatography, calmodulin-Sepharose 4B chromatography and Superose 12 chromatography. Moreover, calmodulin-dependent phosphodiesterase, and independent phosphodiesterase were separated from crude lens extract using DEAE-cellulose column. The calmodulin-dependent phosphodiesterase was purified 4500-fold with a 0.7% yield; it was a dimer formed with two single polypeptides of 59K as the molecular weight. The enzyme had a higher affinity for cyclic GMP than for cyclic AMP, and functioned at calcium ion concentration above 10(-6) M in the incubation mixture. W-7 as calmodulin antagonist indirectly inhibited the enzyme activity and nifedipine as calmodulin-dependent phosphodiesterase antagonist directly inhibited the enzyme activity. These results suggest that an appearance of calmodulin-dependent phosphodiesterase activity depends on the interrelation between the calcium ion and calmodulin in the lens.     

Cytochemical localization of guanylate cyclase in photoreceptor cells of the mouse

Background: Light-stimulated excitation causes a decrease of the cGMP concentration in vertebrate photoreceptor cells. The cGMP content is restored by the catalytic action of a guanylate cyclase (EC 4.6.1.2). Methods: The spatial distribution of guanylate cyclase was determined cytochemically in rod visual cells of the mouse. Results: In retinal tissue of the mouse guanylate cyclase was found throughout the photoreceptor cells, in the outer and the inner segments, and was especially prominent in the cilia and in elongations of cilia extending into the outer segments. A reaction product of adenylate cyclase (EC 4.6.1.1) could not be demonstrated in vertebrate rod outer segments. Conclusion: The relatively high amount of guanylate cyclase in the inner segments and the cilia may contribute — at least in part — to the actual concentration and the time course of concentration changes of the cGMP concentration in rod outer segments.     

Proceedings: Physiology and enzymology of frog photoreceptor membranes

We have developed a convenient and reproducible assay for observing visual excitation and dark adaptation in suspensions of isolated bullfrog rod outer segments. These outer segments can be separated from other contaminating membranes using gentle procedures which do not destroy their physiological activity. Rhodopsin phosphorylating activity and cyclic nucleotide phosphodiesterase remain with these outer segments; ATPase and adenyl cyclase generally thought to be associated with outer segments are not present. In the light-induced rhodopsin phosphorylation reaction low levels of illumination cause the incorporation of more than one phosphate per rhodopsin bleached. This is the only reaction yet characterized in this system which involves an obvious amplification mechanism.     

Atrial natriuretic peptide (ANP), guanylate cyclase, and intraocular pressure in the rabbit eye

Synthetic rat atrial natriuretic peptide (ANP) was examined for effects on guanylate-and on adenylate cyclase activity in ciliary process homogenates and for effects on intraocular pressure in the albino rabbit eye. Ciliary process guanylate cyclase was associated predominantly with the particulate fraction and was partially activated by ANP (EC50, approximately 1 nM) relative to a maximal dose of Na Nitroprusside (2 uM), whereas particulate adenylate cyclase (basal as well as forskolin-stimulated activity) showed no responses to ANP at doses up to 0.3 uM. Particulate cAMP phosphodiesterase activity was stimulated by low doses of cGMP (1-5 uM) in ciliary processes. Thus, ANP, acting via guanylate cyclase, has the potential to regulate phosphodiesterase activity and indirectly decrease cAMP levels in membranes derived from ciliary processes. Intravitreous injection of ANP (2-4 ug/eye) caused a small decrease (1-1.5 mm Hg) in intraocular pressure measured 16-24 hours after injection but the pressure had returned to normal by 40 hours. The findings demonstrate regulation of biochemical and pharmacological responses by ANP in the albino rabbit eye suggesting that this peptide may play a physiological role in secretory functions of ciliary processes.     

Abnormal guanosine 3', 5'-monophosphate during photoreceptor degeneration in the inherited retinal disorder of C3H/HeJ mice.

Elevated levels of guanosine 3'-5'-monophosphate (cyclic GMP) are associated with photoreceptor degeneration in the retina of C3H/HeJ mice. This abonormality has been shown to result from a deficiency in the activity of a receptor-specific cyclic GMP phosphodiesterase. The apparent susceptibility of photoreceptor cells to an abnormality in cyclic GMP metabolism suggests that cyclic GMP may play a role in photoreceptor function which is essential to the viability of these specialized cells.     

Effects of diamide on cyclic nucleotide levels in rat retina

The concentrations of guanosine 3',5' monophosphate (cyclic GMP) and adenosine 3',5' monophosphate (cyclic AMP) were measured in rat retinas incubated under control conditions and in retinas incubated with diamide, a relatively specific glutathione oxidant. Retinas incubated with either glucose or pyruvate as the substrate for 30 min in the dark contained about 50 picomoles cyclic GMP/mg protein and 7 picomoles cyclic AMP/mg protein. Light-exposed control retinas contained about 70% less cyclic GMP (14.4 picomoles/mg protein) and 20% less cyclic AMP (5.4 picomoles/mg protein). Addition of diamide to the incubation medium at concentrations between 0.1 and 1.0 mM produced a concentration-dependent decrease in the dark level of cyclic GMP, but did not affect its concentration in the light or the concentration of cyclic AMP in dark-maintained and light-exposed retinas. The major effect of diamide was to reduce the normal dark-light difference in the concentration of cyclic GMP. Oxidizing conditions thus appear to alter selectively the light-sensitive compartment of retinal cyclic GMP.