Cyclic GMP metabolism in macrophages. I. Regulation of cyclic GMP levels by calcium and stimulation of cyclic GMP synthesis by NO-generating agents

Levels of guanosine 3′,5′-cyclic monophosphate (cGMP) were determined by radioimmunoassay in adherence-purified, oil-induced guinea pig peritoneal exudate macrophages, after extraction of the cells with perchloric acid, purification on Dowex AG1-X8, and acetylation. We found that: (i) Basal cGMP levels were strictly dependent on the concentration of extracellular Ca²⁺ (0.33 ± 0.03 pmol/mg macrophage protein in Ca²⁺-free medium and 2.49 ± 0.42 pmol/mg in 1.8 mM Ca²⁺). (ii) The stimulatory effect of Ca²⁺ on cGMP levels was prevented by tetracaine. (iii) The cGMP content of macrophages was not elevated by incubation with the ionophore A23187 at extracellular Ca²⁺ concentrations varying between 0 and 1.8 mM. (iv) Macrophage cGMP levels were increased markedly (up to 40-fold) by incubation of the cells with the nitric oxide (NO)-generating agents, sodium azide, hydroxylamine, sodium nitrite, and sodium nitroprusside. (v) Stimulation of cGMP accumulation by NO-generating agents occurred within 30 sec, was Ca²⁺-independent, and developed in the presence and absence of the phosphodiesterase inhibitor, isobutyl-methylxanthine. (vi) A minimal elevation in the macrophage cGMP level (less than 2-fold) was induced by ascorbic acid but no significant increases were induced by the following agents, found effective in other cells: serotonin, acetylcholine, carbamylcholine, phorbol myristate acetate, arachidonic acid, Superoxide dismutase, and nitrate reductase.     

Direct evidence that bacterial lipopolysaccharides elevate cyclic GMP levels in rat fetal liver cells

We have previously shown that crude bacterial lipopolysaccharide (LPS) preparations markedly increase cGMP levels in rat fetal liver cells in a time- and dose-dependent manner. To provide evidence that this effect was due to LPS and not an impurity in the preparations, three series of experiments were undertaken. First, LPS was prepared from Escherichia coli 055:B5 cells and its cGMP potency assessed at various stages of purification; second, the cGMP activity of three highly purified LPS preparations of known chemical structure was measured, and third, a well characterized LPS was broken into its lipid A and polysaccharide fractions and the cGMP activity of each fraction determined. The results showed that the cGMP stimulatory activity in E. coli 055:B5 cells co-purified in a parallel fashion with the LPS molecule derived from those cells, that the three chemically defined, highly purified LPS preparations were all very potent stimulators of cGMP levels, and that the ability to increase cGMP levels of lipid A prepared from a highly purified LPS was comparable in potency to the intact LPS, whereas the polysaccharide portion of the molecule was without activity. These findings indicate that the cGMP effect of LPS preparation is due to LPS and not a contaminant and that the activity resides within the lipid A moiety of the molecule.     

Regulation of cyclic GMP levels in nerve tissue

In rat superior cervical ganglia the regulation of cyclic GMP (cGMP) formation does not involve muscarinic or adrenergic transmitters or receptors. Marked increases in cGMP content during preganglionic axonal stimulation by electric currents, elevated K+, or drugs that cause transmitter release are unaffected by muscarinic and adrenergic receptor blockade. However, the cGMP response does require Ca2+ and intact preganglionic axonal terminals. Two possibilities exist: either cGMP accumulates in the preganglionic nerves or a noncholinergic, nonadrenergic transmitter activates guanylate cyclase in postsynaptic structures. Sodium azide and nitroprusside cause cGMP accumulation in denervated ganglia, which indicates that postsynaptic structures are capable of forming cGMP. In pineal glands elevated [K+]o releases [3H]norepinephrine and causes cGMP accumulation, which suggests a relationship between the two responses and the possibility that cGMP accumulation is involved in autoinhibition of transmitter release. The finding that phentolamine, alpha-adrenergic receptor antagonists, prevent the cGMP response to K+ is compatible with this review. However, clonidine, an alpha-receptor agonist, depresses norepinephrine release but has no effect on pineal gland cGMP. Conversely, large increases in pineal gland cGMP produced by nitroprusside do not affect K+-evoked norepinephrine release. For these reasons it is not possible to relate cGMP to the auto-inhibition of [3H]norepinephrine release that is mediated by prejunctional alpha-adrenergic receptors.     

Effects of intracellular cyclic AMP and cyclic GMP levels on DNA synthesis of young-adult rat hepatocytes in primary culture.

Possible roles of dibutyryladenosine 3',5'-cyclic monophosphate (cAMP) and dibutyryl-guanosine 3',5'-cyclic monophosphate (cGMP) in regulation of hepatocyte DNA synthesis were examined using primary cultures of young-adult rat hepatocytes maintained in arginine-free medium. Throughout the experimental period, nonparenchymal cells were hardly observed in the selective medium. When epidermal growth factor (EGF) was added to the cultures, a transient increase in the intracellular cAMP level preceded the elevation of hepatocyte DNA synthesis. EGF-stimulated hepatocyte DNA synthesis was remarkably enhanced by the elevation of the intracellular cAMP level induced by treatment with cAMP alone or a combination of cAMP and theophylline, an inhibitor of cyclic nucleotide phosphodiesterase. Furthermore, the early elevation of intracellular cAMP alone, which was induced by treatment with the combination of cAMP and theophylline, caused a remarkable increase in hepatocyte DNA synthesis. On the other hand, addition of EGF to the cultures caused a rapid decrease in the intracellular cGMP level followed by an increase in hepatocyte DNA synthesis. EGF-stimulated hepatocyte DNA synthesis was severely suppressed or completely inhibited by the elevation of the intracellular cGMP level induced by treatment with cGMP alone or a combination of cGMP and dipyridamole, a specific inhibitor of cGMP phosphodiesterase. These findings indicate that cAMP and cGMP act oppositely on the regulation of DNA synthesis of young-adult rat hepatocytes in primary culture: cAMP plays a positive role, whereas cGMP plays a negative role. Also it is strongly suggested that an early elevation of the intracellular cAMP level is essential for the onset of DNA synthesis in hepatocyte primary cultures.     

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.     

Immunologic characterization of the photoreceptor outer segment cyclic GMP phosphodiesterase

High affinity (KD approximately 1 X 10(-9) M) monoclonal antibodies (ROS-1 and ROS-2) were prepared to bovine photoreceptor outer segment cGMP phosphodiesterase. ROS-1 immunoadsorbed greater than 95% of the cGMP phosphodiesterase activity from a detergent-solubilized bovine retina extract while ROS-2 immunoadsorbed only a subfraction of the same activity. Sodium dodecyl sulfate gel analysis of these immunoadsorbates demonstrated that ROS-1 and ROS-2 specifically adsorbed only peptides that comigrated with purified rod outer segment phosphodiesterase. Limited trypsin digestion of purified rod outer segment phosphodiesterase greatly reduced its affinity for ROS-1 but not ROS-2. When a crude heat-stable inhibitor fraction was added back to the activated enzyme, the affinity for ROS-1 was restored, suggesting that the inhibitor was necessary for ROS-1 binding. ROS-1 but not ROS-2 was found to inhibit cGMP phosphodiesterase which had been activated either by dilution or guanyl nucleotide. The inhibitory property of ROS-1 may provide a useful probe for directly studying the effects of this phosphodiesterase on the phototransduction response in the retina. Sodium dodecyl sulfate gel analysis demonstrated that the ROS-1 immunoadsorbates from mammals, fish, and amphibia contained peptides of similar mobility. Immunocytochemistry performed with ROS-1 and fluorescein isothiocyanate-conjugated rabbit anti-mouse IgG localized the antigenic determinant to both rod and cone outer segments suggesting the presence of an antigenically similar phosphodiesterase in both types of photoreceptors.     

Effect of endogenous LH secretion on ovarian cyclic AMP and cyclic GMP levels in the rat

Injection of LH (2 and 10 μg) into proestrus rats increased ovarian cyclic AMP levels and concomitantly decreased the levels of cyclic GMP. When injected into diestrus rats, cyclic AMP increases were even greater, whereas cyclic GMP levels were not significantly different from controls receiving saline injections. Ovarian cyclic nucleotide levels were also examined on different days of the cycle. On the afternoon of proestrus (1700 h), the time when circulating levels of LH are at their maximum, the concentration of cyclic AMP showed a moderate but insignificant increase. At the same time, cyclic GMP levels were significantly decreased. An inverse relation between cyclic AMP and cyclic GMP levels was seen on each day of the cycle. When rats were injected with pentobarbital (35 mg/kg) on the afternoon of proestrus (1300 h) to block the LH surge, the expected increases in ovarian cyclic AMP and decreases in cyclic GMP were effectively blocked. These results indicate that ovarian cyclic AMP and cyclic GMP levels are regulated by circulating LH. The apparent differences in direction of nucleotide response to LH, suggest divergent roles for the nucleotides in ovarian function.     

Inhibition of platelet aggregation by 6-keto-PGE1; lack of an effect on cyclic GMP levels

The effects of 6-keto-PGE₁ on aggregatory responses to arachidonic acid (AA), adenosine diphosphate (ADP) and collagen were studied in human platelet-rich plasma (PRP). In addition, experiments were carried out to determine if these effects correlate with changes in platelet cyclic AMP and cyclic GMP levels. 6-Keto-PGE₁ incubated in PRP produced dose-related increases in platelet cyclic AMP levels whereas platelet cyclic GMP levels were unchanged. Control aggregations induced by AA and ADP did not alter cyclic AMP and cyclic GMP levels whereas control aggregations induced by collagen elevated cyclic GMP levels while cyclic AMP levels were unchanged. 6-Keto-PGE₁ produced a dose-dependent inhibition of platelet aggregation induced by AA, ADP and collagen and this inhibition correlated with a dose-related increase in cyclic AMP levels. Since 6-keto-PGE₁ does not consistently alter cyclic GMP levels in human PRP, the present data support previous studies suggesting that 6-keto-PGE₁ produces inhibition of platelet aggregation through the stimulation of cyclic AMP accumulation.     

Effect of sodium ions on cyclic AMP and cyclic GMP levels in mouse parotid acini

The ability of the β-adrenergic agonist, isoproterenol, to elevate intracellular levels of cyclic-AMP (c-AMP) and cyclic GMP (c-GMP) in mouse parotid acini was dependent upon the extracellular sodium concentration. In the absence of extracellular sodium isoproterenol-stimulated c-GMP and c-AMP levels were significantly reduced; carbachol-stimulated c-GMP levels were not affected. Monensin, a sodium ionophore, mimicked the effects of isoproterenol in elevating c-GMP levels; this effect was abolished in the absence of extracellular sodium. Monensin did not mimic the effects of isoproterenol in elevating c-AMP levels. The data presented suggests that sodium ions may play a role in β-adrenergic regulation of cyclic nucleotide levels in mouse parotid gland and that the mechanisms involved in regulation of c-AMP and c-GMP levels appear to be different.     

The action of tomicide on macromolecular synthesis in bacterial cells]

The study of the rate of incorporation of labeled precursors for nucleic acids and protein into Staphylococcus aureus 209 P cell fraction, insoluble in trichloroacetic acid, has revealed that in the presence of tomicide in the medium in a dose of 1 MCI (600 micrograms/ml) the synthesis of DNA in inhibited rapidly and almost completely (by 90%). The inhibition of the rate of incorporation of 3H-thymidine into the cells of staphylococcal culture by tomicide directly correlates with the concentration of the preparation within the range 100-600 micrograms/ml, the inhibition of the synthesis of RNA and protein being less pronounced than the inhibition of the synthesis of DNA.     

Regulation of intracellular cyclic GMP levels in olfactory sensory neurons

Cyclic AMP is the primary second messenger mediating odorant signal transduction in mammals. A number of studies indicate that cyclic GMP is also involved in a variety of other olfactory signal transduction processes, including adaptation, neuronal development, and long-term cellular responses in the setting of odorant stimulation. However, the mechanisms that control the production and degradation of cGMP in olfactory sensory neurons (OSNs) remain unclear. Here, we investigate these mechanisms using primary cultures of OSNs. We demonstrate that odorants increase cGMP levels in intact OSNs in vitro. Different from the rapid and transient cAMP responses to odorants, the cGMP elevation is both delayed and sustained. Inhibition of soluble guanylyl cyclase and heme oxygenase blocks these odorant-induced cGMP increases, whereas inhibition of cGMP PDEs (phosphodiesterases) increases this response. cGMP PDE activity is increased by odorant stimulation, and is sensitive to both ambient calcium and cAMP concentrations. Calcium stimulates cGMP PDE activity, whereas cAMP and protein kinase A appears to inhibit it. These data demonstrate a mechanism by which odorant stimulation may regulate cGMP levels through the modulation of cAMP and calcium level in OSNs. Such interactions between odorants and second messenger systems may be important to the integration of immediate and long-term responses in the setting odorant stimulation.     

Evidence against direct involvement of cyclic GMP or cyclic AMP in bacterial chemotactic signaling.

Defects in phosphotransferase chemotaxis in cya and cpd mutants previously cited as evidence of a cyclic GMP or cyclic AMP intermediate in signal transduction were not reproduced in a study of chemotaxis in Escherichia coli and Salmonella typhimurium. In cya mutants, which lack adenylate cyclase, the addition of cyclic AMP was required for synthesis of proteins that were necessary for phosphotransferase transport and chemotaxis. However, the induced cells retained normal phosphotransferase chemotaxis after cyclic AMP was removed. Phosphotransferase chemotaxis was normal in a cpd mutant of S. typhimurium that has elevated levels of cyclic GMP and cyclic AMP. S. typhimurium crr mutants are deficient in enzyme III glucose, which is a component of the glucose transport system, and a regulator of adenylate cyclase. After preincubation with cyclic AMP, the crr mutants were deficient in enzyme II glucose-mediated transport and chemotaxis, but other chemotactic responses were normal. It is concluded that cyclic GMP does not determine the frequency of tumbling and is probably not a component of the transduction pathway. The only known role of cyclic AMP is in the synthesis of some proteins that are subject to catabolite repression.     

Regulation of cyclic AMP and cyclic GMP levels by adrenocorticotropic hormone in cultured neurons

The effect of adrenocorticotropic hormone (ACTH) on the intracellular concentration of cyclic nucleotides was studied in cultures of neurons from embryonic chick cerebral hemispheres. Incubation of neurons with ACTH(1-24) in the presence of phosphodiesterase inhibitor isobutylmethylxanthine resulted in a sustained increase in cyclic AMP while rise in cyclic GMP level was transient. The values obtained for half-maximal stimulation were 0.5 microM and 0.03 nM for cyclic AMP and cyclic GMP respectively. Concomitantly, ACTH(1-24) stimulated guanylate cyclase activity (half-maximal stimulation at 0.02 nM). These results suggest the existence of two distinct populations of ACTH receptors in neurons and provide the first evidence that cyclic GMP does mediate the action of ACTH in neurons.     

The effect of cyclic GMP on phosphofructokinase from rat tissues.

In view of the recently proposed hypothesis of biologic regulation through opposing influences of cyclic AMP and cyclic GMP, and since cyclic AMP is a well-known allosteric activator of phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11), the effect of cyclic GMP on the activity of this enzyme from several rat tissues was investigated. It was found that cyclic GMP exerted an inhibitory effect on the activity of rat heart and skeletal muscle phosphofructokinase. This effect was most pronounced under conditions in which the enzyme was partially inhibited by ATP or by citrate. Cyclic GMP also antagonized the deinhibitory action of cyclic AMP and other allosteric activators, such as glucose 1,6-bisphosphate or AMP, on the ATP or citrate-inhibited heart or muscle phosphofructokinase. In contrast to the heart and skeletal muscle phosphofructokinase, the adipose-tissue enzyme was not affected by cyclic GMP to any significant degree. The antagonistic action of cyclic GMP to the activation of heart-phosphofructokinase, may suggest a mechanism by which the activity of phosphofructokinase is synchronized with the activity of glycogen phosphorylase, as a result of acetylcholine action in heart, to achieve a decrease in total glycogenolysis and glycolysis.     

Substance P increases cyclic GMP levels on coronary postcapillary venular endothelial cells

The vasodilating effect of substance P (SP) at the microvascular level is endothelium-dependent. In the present study we evaluated whether SP activates nitric oxide (NO) production by venular endothelial cell. We evaluated NO activation by measuring cyclic GMP levels in cultured endothelial cells isolated from coronary postcapillary venules of bovine origin (CVEC). Our results indicate that 5 min exposure of CVEC to 10 nM SP doubled basal cyclic GMP levels. Cell treatment with the NO synthase inhibitor L-NMMA reduced the basal levels of cyclic GMP and abolished the effect of SP but did not modify the increase in cyclic GMP in response to exogenous NO. These data indicate that a) microvascular endothelium responds in an autocrine fashion to NO with increased cyclic GMP levels, b) SP activates cyclic GMP pathway through NO production.