Immunohistochemical localization of cyclic GMP in aggregating Polysphondylium violaceum.

Using indirect immunofluorescence technique, it has been possible to localize cyclic GMP in Polysphondylium violaceum cells. The bound cyclic nucleotide is localized throughout the cell during early stages, however, this staining increases and there is marked localization of cyclic GMP in the nuclear areas of the cells when aggregation is in full swing. Over 90% of the cells exhibited intense nuclear staining by 6 h and this decreased to less than 10% by 10 h.     

Immunohistochemical Localization of Cyclic GMP in Aggregating Polysphondylium violaceum

Using indirect immunofluorescence technique, it has been possible to localize cyclic GMP in Polysphondylium violaceum cells. The bound cyclic nucleotide is localized throughout the cell during early stages, however, this staining increases and there is marked localization of cyclic GMP in the nuclear areas of the cells when aggregation is in full swing. Over 90% of the cells exhibited intense nuclear staining by 6 h and this decreased to less than 10% by 10 h.     

Characterization of immunofluorescent cyclic GMP-positive fibres in the central nervous system.

The cyclic GMP immunofluorescent fibres in the rat central nervous system have been characterized as processes of fibrous astrocytes, on the basis of distribution and similarity to the localization of glial fibrillary acidic protein. The neuroglial localization of the nucleotide is discussed together with the surprising observation that these cyclic GMP positive fibres are absent from the central nervous system of the adult mouse.     

Ultrastructural localization of cyclic GMP and cyclic AMP in rat striatum

The subcellular localization of cyclic GMP and cyclic AMP in the rat caudate-putamen has been studied using horseradish peroxidase immunocytochemistry. Both of the putative neurotransmitter second messengers were visualized in neurons and glial cells at light microscopic resolutions, but not all cells of either category gave detectable staining. This was confirmed at the ultrastructural level where both stained and unstained elements of the same cell type were found within the same field. A striking variation was seen in cyclic nucleotide staining intensity within individual neural and glial cells. Both of the cyclic nucleotides were detected within postsynaptic terminal boutons and within astroglial processes. Cyclic GMP postsynaptic staining was stronger than glial staining, whereas the localization pattern was reversed for cyclic AMP. The synaptic localization of cyclic AMP and cyclic GMP immunoreactivity adds support to the idea that these compounds have an influential role in synaptic function within the striatum.     

A method for measuring relative changes in guanosine 3'':5''-cyclic monophosphate in mouse neuroblastoma cells on muscarinic cholinergic stimulation.

A convenient, inexpensive assay was developed for measuring relative changes in cyclic GMP in whole mouse neuroblastoma cells (clone NIE 115) based on labelling the cellular GTP pool with [8(-3)H]guanine. The time course of cell labelling and the distribution of radioactivity among possible products were studied; GTP is the only major labelled species. Radioactive cyclic GMP produced from the radioactive GTP on cell stimulation is isolated by column chromatography nad its identity has been rigorously established by paper chromatography and ion-exchange chromatography. The assay was used to study the time course of the cyclic GMP changes that occur after stimulation of neuroblastoma cells with carbamoylcholine and the dependence of the cyclic GMP changes on the carbamoylcholine concentration. The assay gives results comparable with those obtained by using a radioimmunoassay for cyclic GMP and should be applicable to other whole-cell and tissue-slice systems.     

Cyclic GMP levels and membrane current during onset, recovery, and light adaptation of the photoresponse of detached frog photoreceptors

We have used a preparation of isolated, intact rod photoreceptors to correlate the effects of flash illumination on the intracellular cyclic GMP content and the membrane current. We find that the recovery of cyclic GMP levels after brief flash illumination requires approximately twice as much time as the recovery of the membrane current. In contrast, the subsecond kinetics of the cyclic GMP response to light are faster than the kinetics of membrane current suppression. Both cyclic GMP and the membrane current show graded responses to a wide range of flash intensities; however, in a low Ca2+-Ringer's solution, dim flashes can trigger a decrease in cyclic GMP concentration with no corresponding decrease in membrane current. These results suggest that either other factors can regulate the membrane current, or that measurements of total cellular cyclic GMP do not accurately reflect dynamic changes in cyclic GMP concentration in the vicinity of the light-regulated channel. Changes in cyclic GMP concentration in the presence of background illumination exhibit adaptational behavior similar to that observed in a light-adapted photoresponse: acceleration in the response kinetics and a decrease in response amplitude. That this result is observed in rods depleted of internal Ca2+ suggests a Ca2+-independent mechanism by which background illumination can accelerate the cyclic GMP response.     

Kinetics of the Hydrolysis of 8-Bromo-Cyclic GMP by the Light-Activated Phosphodiesterase of Toad Rods

The hypothesis that cyclic GMP is the internal transmitter of retinal rod phototransduction, when combined with the observations that 8-bromo-cyclic GMP opens the cyclic GMP-dependent outer segment conductance and that rods into which 8-bromo-cyclic GMP has been injected still respond to light, predicts that the light-activated phosphodiesterase (EC 3.1.4.17) must catalyze the hydrolysis of 8-bromo-cyclic GMP. This hypothesis was tested by measuring light-activated toad rod disk membrane phosphodiesterase with a pH assay technique. Phosphodiesterase-catalyzed hydrolysis of 8-bromo-cyclic GMP was confirmed: at pH 8.0, total proton production after flash activation was identical to total amount of 8-bromo-cyclic GMP added as substrate. Photoactivated phosphodiesterase was remarkably less efficient in catalyzing the hydrolysis of 8-bromo-cyclic GMP than of cyclic GMP: Vmax for 8-bromo-cyclic GMP was 0.063 M/M rhodopsin/s, whereas that for cyclic GMP was 11 M/M rhodopsin/s--170 times greater. The Km for 8-bromo-cyclic GMP was 160 microM, and for cyclic GMP, 590 microM. 8-bromo-cyclic GMP competitively inhibited phosphodiesterase-catalyzed hydrolysis of cyclic GMP with a Ki of 1.2 mM. Complete reaction progress curves were analyzed for obedience to Michaelis-Menten kinetics: cyclic GMP hydrolysis, 8-bromo-cyclic GMP hydrolysis, and cyclic GMP hydrolysis in the presence of 8-bromo-cyclic GMP as competitive inhibitor were found to follow the integrated form of the Michaelis-Menten equation over the time course of the reactions, assuming phosphodiesterase was activated as a step. The kinetic parameters extracted from reaction progress curves were consistent with those derived from analysis of the initial velocity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of dose and time after administration of 4-isopropyl-2,6,7-trioxa-1-phosphabicyclo (2,2,2)octane-1-oxide (IPTBO) on cyclic nucleotide concentrations in mouse cerebellum

The concentrations of cyclic AMP and cyclic GMP in the mouse cerebellum after intracerebroventricular administration of a range of doses of IPTBO have been studied with particular interest in the temporal changes after injection. A non typical dose relationship was observed. After the lowest and non-convulsive dose used (0.06 μg/animal) cyclic AMP levels decreased and cyclic GMP levels increased within 1 min, but after higher doses cyclic AMP and cyclic GMP levels were both raised. At three different convulsive doses of IPTBO there were increased levels of cyclic AMP with time which were more apparent in convulsing animals. Raised levels of cyclic GMP however, were not so influenced by convulsions. The results suggest that (1) the immediate decrease in cyclic AMP and the immediate increase in cyclic GMP may play a part in the mechanism of action of IPTBO—possibly by triggering convulsions and (2) there is an increase in cyclic AMP in response to, or because of, the convulsions. It is concluded that time after treatment and time into convulsions are critical when studying cyclic nucleotide changes, particularly for cyclic AMP and that such factors may explain conflicting observations with respect to this nucleotide.     

Ca2+-dependent changes in cyclic GMP levels are not correlated with opening and closing of the light-dependent permeability of toad photoreceptors

We have measured the levels of 3',5'-guanosine monophosphate (cyclic GMP) in isolated retinas from toad to investigate their correlation to the opening and closing of the light-dependent permeability of photoreceptors. When Ca2+-induced changes in cyclic GMP concentration are compared with the Ca2+-induced changes in the permeability of photoreceptor light-dependent channel, four quantitative dissimilarities are noted. First, when extracellular Ca2+ ([Ca2+]o) is reduced from normal physiological levels to between 10(-6) and 10(-7) M, the light-dependent permeability is increased, but cyclic GMP levels are not significantly changed. Second, when [Ca2+]o is increased from 1.8 to 20 mM, the light-dependent permeability is suppressed, but cyclic GMP levels are decreased by only 10-15%, about one-quarter the decrease that can be obtained with bright illumination. Third, when [Ca2+]o is increased from 10(-8) M to 20 mM, the light-dependent permeability is closed rapidly, but the cyclic GMP decrease is slow. Fourth, when [Ca2+]o is lowered to 10(-8) M, the sensitivity of the light-dependent permeability to steady illumination is decreased by three to four orders of magnitude, but the sensitivity of the light-dependent decrease in cyclic GMP is not significantly affected. These observations indicate that there is no simple correlation between cyclic GMP levels and the permeability of the light-dependent channels and that Ca2+ can affect the conductance in the absence of changes in cyclic GMP content.     

Extracellular localization of cyclic GMP in the house cricket male accessory reproductive gland and its fate in mating

The male accessory reproductive gland (ARG) of the house cricket, Acheta domesticus (L.), contains an exceedingly high concentration of cyclic GMP, about 1,000 pmol/mg protein. Immunofluorescent localization and radioimmunoassay measurements show that cyclic GMP is concentrated in a small number of tubules. It accumulates in the tubule lumina where it is protected from degradation by phosphodiesterases. Cyclic GMP is secreted by the ARG and is incorporated into spermatophores. Over 80% of spermatophore cyclic GMP is found in the handle-capillary tube, a thin conduit through which sperm pass during transfer to the female. The concentration of cyclic GMP in the insemination fluid is about 20 microM but does not appear to be specifically associated with the sperm. Cyclic GMP enters the female spermatheca during insemination but disappears rapidly. Physiological effects of cyclic GMP on sperm were not observed nor was an effect of cyclic GMP observed on egg laying by mated females. Cyclic AMP was localized on sperm flagella in the spermatophore and in the spermatheca. These studies indicate that cyclic nucleotides have important roles in insect reproduction and that the house cricket is a good model for elucidating these functions.     

Deranged metabolism of cyclic nucleotides in liver diseases

To clarify the factor(s) responsible for changes in the plasma cyclic GMP concentration in liver diseases, we measured the plasma levels of cyclic GMP, along with cyclic AMP, in various clinical stages of chronic liver diseases and acute hepatitis. The level of cyclic GMP was found to increase significantly in the early stage of acute hepatitis, in the decompensated stage of liver cirrhosis, and in malignant diseases. In the former two states, it is postulated that decreased hepatic mass is responsible for the changes in the plasma cyclic GMP concentration. The retention rate of indocyanin green (ICGR15) was highly correlated with the plasma cyclic GMP level. The result suggests that the determination of plasma cyclic GMP is useful as an index of the reserve function of the liver in disease states.     

Studies on the specificity of immunohistochemical techniques for cyclic AMP and cyclic GMP.

Immunohistochemical studies employing antibodies against cyclic nucleotides indicate that cyclic AMP and cyclic GMP are localized to distinct subcellular sites. These antibodies, however, cross-react weakly with noncyclic nucleotides (eg. ATP, GTP), and therefore we investigated the speficity of the immunohistochemical technique. Slides of fetal nuclei exposed to gaseous nitrous acid demonstrated reduced immunofluorescence. The slides were then incubated with cyclic and noncyclic nucleotides, and restoration of distinct cyclic AMP and cyclic GMP staining pattern was achieved only with appropriate cyclic nucleotides. Antibodies that were used have a greater affinity for acetylated derivatives of cyclic nucleotides. By using a gas phase technique, tissue slices were acetylated and immunohistochemical staining intensity was compared with the effect of acetylation on antibody affinity for various nucleotides. Acetylation greatly increased affinity of cyclic AMP antibody for cyclic AMP but not other nucleotides, and greatly intensified cyclic AMP staining. Acetylation moderately increased affinity of cyclic GMP antibody for cyclic GMP, and moderately intensified cyclic GMP staining. Conclusion: Both nitrous acid and acetylation studies support the specificity of the immunohistochemical method for cyclic nucleotides.     

Cyclic GMP regulates cromakalim-induced relaxation in the rat aortic smooth muscle: role of cyclic GMP in K(ATP)-channels.

Recent studies have shown that nitric oxide (NO) modulates K+-channel activity which play an important role in controlling vascular tone. The formation of cyclic guanosine 3',5'-monophosphate (cyclic GMP) has also been recognized to be associated with the vasodilatory effect of NO. Both cyclic GMP and NO increase whole-cell K+-current by activating Ca2+-activated K+-channels (K(Ca)-channels). Here, we show evidence that activators of soluble guanylyl cyclase sodium nitroprusside or 3-morpholino-sydnonimine (SIN-1), and an analogue of cyclic GMP 8-bromo-cyclic GMP enhance the relaxation induced by cromakalim which is blocked by glibenclamide (a specific inhibitor of ATP-sensitive K+-channels [K(ATP)-channels]), and partially attenuated by methylene blue (an inhibitor of cyclic GMP formation). However, this is not due to the increase of cyclic GMP level by cromakalim itself because the relaxation induced by cromakalim is not associated with the changes of cyclic GMP level formed in the aortic smooth muscle. Thus, it is most likely that cyclic GMP also modulates activity of K(ATP)-channels, in addition to K(Ca)-channels, in the rat aorta.     

A microassay for guanosine 3',5'-monophosphate phosphodiesterase activity

The activity of cyclic GMP phosphodiesterase was determined using a three step procedure. In the first step, cyclic GMP phosphodiesterase catalyzes the conversion of cyclic GMP to 5′-GMP. In the second step, a known amount of ATP and guanylate kinase are incubated with the 5′-GMP formed in the first step. The amount of ATP which remains is inversely related to the amount of 5′-GMP formed. In the third step, the concentration of ATP is measured using the firefly luciferin-luciferase technique. The validity of the assay is confirmed by its ability to show the linearity of the cyclic GMP phosphodiesterase reaction with respect both to time of incubation and concentration of tissue. It is capable of detecting less than 5 pmoles of 5′-GMP in 150 μl, and can be used to measure cyclic GMP phosphodiesterase activity in a supernatant fraction of rat cerebrum which contains less than 25 ng of protein. It has been used to determine the activity and properties of cyclic GMP phosphodiesterase in unpurified supernatant and particulate fractions of several tissues of the rat, as well as in highly purified fractions of rat caudate nucleus.     

Nitroarginine does not inhibit lysophosphatidylcholine (LPC)-induced vascular relaxation and accumulation of cyclic GMP.

Lysophosphatidylcholine (LPC) is a potent endothelium-dependent vascular smooth muscle relaxant. The possibility that its action is mediated through endothelium-derived nitric oxide (EDNO), although suggestive, has not been proven. Both lysophosphatidylcholine and endothelium-derived nitric oxide relax by activating guanylate cyclase to form cyclic GMP. Based on the finding that EDNO formation is inhibited by NNA (N-omega-nitro-L-arginine), we followed cyclic GMP changes in bovine intrapulmonary arteries with LPC after incubation with NNA. Inhibition of cyclic GMP by LPC following NNA exposure would be suggestive of the production of EDNO by LPC. However, while NNA significantly inhibited accumulation of cyclic GMP after exposure to the calcium ionophore A23187 which releases EDNO, NNA failed to inhibit LPC-induced accumulation of cyclic GMP. The results indicate that LPC relaxes vascular smooth muscle through a non NO-mediated pathway.     

ALTERATIONS IN ACETYLCHOLINE SYNTHESIS AND CYCLIC NUCLEOTIDES IN MILD CEREBRAL HYPOXIA

In order to elucidate changes accompanying mild cerebral hypoxia, the synthesis of the neurotransmitter acetylcholine and the concentrations of cyclic AMP and cyclic GMP have been compared to changes in brain lactate in the forebrain of mice made mildly hypoxic. Both histotoxic hypoxia (injection of KCN) and anemic hypoxia (injection of NaNO₂) were studied. Acetylcholine synthesis was followed by a double-label technique using [U-¹⁴C] glucose and [²H₄] choline. A 43%, decrease in the synthesis of acetylcholine from [U-¹⁴C]glucose and an 80% increase of the level of cyclic GMP accompanied hypoxia so mild that there were no significant changes in cerebral lactate, or in cyclic AMP (or in AMP: Gibson & Blass , 1976b). Changes in glucose utilization do not account for the decrease in glucose incorporation into acetylcholine. Glucose utilization decreases and then increases with increasing hypoxia, whereas incorporation into acetylcholine decreased with increased hypoxia.     

Effects of parathyroid hormone on cyclic AMP, cyclic GMP, and efflux of calcium in isolated renal tubules.

The effects of parathyroid hormone (PTH) on concentrations of cyclic AMP and cyclic GMP were investigated in isolated renal cortical tubules from hamsters. Efflux of 45Ca from tubules was compared to temporal changes in both cyclic nucleotide concentrations. A rapid increase in cyclic AMP occurred following addition of PTH which was maximal by 1 min but decreased over the next 4 min period. Cyclic GMP concentrations were not significantly altered at 1 min but increased between 1 and 5 min from basal levels. Concentrations of both nucleotides remained significantly elevated from basal levels between 5 and 15 min following PTH. Efflux of 45Ca was increased by PTH with time-course changes closely paralleling changes in cyclic GMP concentrations. Changes in both cyclic AMP and cyclic GMP were related to PTH concentrations of the incubation media and were increased by addition of theophylline. Increasing the calcium concentration from 1 to 3 mM did not significantly alter the effect of PTH on cyclic AMP, however, cyclic GMP concentrations were further increased.     

Cyclic GMP directly regulates a cation conductance in membranes of bovine rods by a cooperative mechanism

Cyclic GMP causes the release of endogenous Ca2+ from rod outer segments, whose plasma membrane has been made permeable, or from isolated discs. Approximately 11,000 Ca2+ ions are released per disc at saturating concentrations of cyclic GMP. The velocity and the amplitude of the release of Ca2+ are dependent on the concentration of cyclic GMP. The maximal rate of the Ca2+ efflux is approximately 7 X 10(4) Ca2+ ions s-1 rod-1. The Ca2+ release by cyclic GMP is independent of light. The activation of the efflux occurred within a narrow range of the cyclic GMP concentration (30-80 microM) and does not obey a simple Michaelis-Menten scheme. Instead, the kinetic analysis of the Ca2+ efflux suggests that a minimum number of 2 molecules of cyclic GMP activates the ion conductance in a cooperative fashion. The release of Ca2+ by cyclic GMP requires a gradient of Ca2+ ions across the disc membrane. If the endogenous Ca2+ gradient is dissipated by means of the ionophore A23187, the release of Ca2+ by cyclic GMP is abolished. Ca2+ is released by analogues of cyclic GMP which are either modified at the 8-carbon position of the imidazole ring or by the deaza-analogue of cyclic GMP. Congeners of cyclic GMP which are modified at the ribose, phosphodiester, or pyrimidine portion of the molecule are ineffective. The hydrolysis of cyclic GMP by the light-regulated phosphodiesterase of rod outer segments is not a necessary condition for the Ca2+ release because 8-bromo-cyclic GMP, a congener resistant to hydrolysis, is a more powerful activator of the release than cyclic GMP itself. Ca2+ release by cyclic GMP is inhibited by organic and inorganic blockers of Ca2+ channels. The l-stereoisomer of cis-diltiazem blocks the release of Ca2+ at micromolar concentrations, whereas the d-form is much less effective. These results suggest that disc membranes contain a cationic conductance which is permeable to Ca2+ ions and which is regulated through the cooperative binding of at least 2 molecules of cyclic GMP to regulatory sites of the transport protein. By this mechanism, subtle changes in the concentration of cyclic GMP could promote large changes in the flux of Ca2+ ions across the disc membrane.     

Oppositional effects of acetylcholine and isoproterenol on isometric tension and cyclic nucleotide concentrations in rabbit atria.

The effects of acetylcholine chloride and isoproterenol on myocardiial cyclic GMP, cyclic AMP and on isometric tension were studied in isolated electrically driven rabbit atria. Acetylcholine (0.5 muM) produced a significant decrease in isometric force that was associated with a significant elevation in atrial cyclic GMP. Cyclic AMP was significantly lowered at 15 seconds after the addition of acetylcholine, but was only slightly decreased at earlier time periods. Both the negative inotropic action and increase in cyclic GMP after addition of acetylcholine were blocked by atropine. Isoproterenol (0.1 muM) produced a significant increase in isometric tension that was associated with a significant elevation in atrial cyclic AMP levels, whereas cyclic GMP levels were not changed. These effects were blocked by practolol. The increases in atrial cyclic GMP and cyclic AMP following addition of acetylcholine and isoproterenol, respectively, preceded the changes in isometric tension in response to these agents. These data support the hypothesis that changes in intracellular levels of cyclic AMP and cyclic GMP may mediate the positive and negative inotropic effects of adrenergic and cholinergic agents.     

Femtomole sensitive radioimmunoassay for cyclic AMP and cyclic GMP after 2'0 acetylation by acetic anhydride in aqueous solution.

The sensitivity of radioimmunoassays for cyclic AMP and cyclic GMP has been markedly improved to readily detect femtomole (10-15) amounts in tissue extracts by acetylating the cyclic nucleotides at the 2'0 position with acetic anhydride. Acetylation of cyclic nucleotides by acetic anhydride in aqueous solution proceeds more rapidly than the hydrolysis of acetic anhydride to acetic acid thus yielding 100% acetylated cyclic nucleotide. 2'0 substituted cyclic nucleotides have greater affinity for the antibody than the parent cyclic nucleotides because the antibody has been made to a protein conjugate coupled at the 2'0 position. This simple acetylation technique makes it possible to measure cyclic AMP and cyclic GMP in minute quantities of tissue without purification or concentration of the sample.