Disorders in the system of cyclic nucleotides in atherosclerosis: cyclic AMP and cyclic GMP content and activity of related enzymes in human aorta

Cyclic AMP and cyclic GMP content and activities of cyclic nucleotide metabolic enzymes were determined in intima and media of atherosclerotic and unaffected human aorta obtained shortly after death due to myocardial infarction. Cyclic AMP content in fatty streaks and atherosclerotic plaques was lower by three- and five-fold, respectively, as compared with uninvolved intima. Cyclic GMP level in atherosclerotic lesions was estimated to be three-fold higher than in grossly normal area. Basal activity of adenylate cyclase in fatty streaks and plaques was two- to six-fold lower than in unaffected intima. Besides, the ability of adenylate cyclase to be stimulated by the stable analogue of prostacyclin, carbacyclin, was suppressed in plaques. Guanylate cyclase activity in fatty streaks was 1.5- to three-fold higher than in normal tissue. The thiol-reducing agent, dithiothreitol, decreased the enzyme activity to normal level, suggesting the oxidative nature of guanylate cyclase activation in the lesion zone. There were no significant changes in cyclic AMP phosphodiestease activity in the regions of the atherosclerotic lesion. Cyclic GMP phosphodiesterase activity in atherosclerotic plaques was two-fold lower than in the intima of unaffected areas. We did not find differences in the content of cyclic nucleotides or related enzyme activities in the media of uninvolved areas of human aorta nor in the media underlying atherosclerotic lesions. Our findings suggest that development of human atherosclerotic lesions is accompanied by dramatic changes in the cyclic nucleotide metabolism featuring gradual hormonal receptor uncoupling from adenylate cyclase, activation of guanylate cyclase in fatty streaks and inhibition of cyclic GMP phosphodiesterase in plaques.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative estimation of lipid-laden cells in atherosclerotic lesions of the human aorta.

The intima of the adult human aorta consists of three sublayers: a muscular layer lying next to the media, a median hyperplastic layer and an innermost connective tissue layer, adjoining the lumen. The cells inhabiting these sublayers were isolated by the method of alcoholic-alkaline dissociation from grossly normal areas, fatty streaks and atherosclerotic plaques. The populations obtained contained cells with different numbers of cytoplasmic inclusions and a number without any. In unaffected intima and in fatty streaks, the cells with lipid inclusions were found predominantly in the outermost intimal layer including the connective tissue and in part of the median hyperplastic layer. In the superficial layer of unaffected intima and the fatty streak, these cells accounted for 15 and 25% of the total cell population, respectively. In the plaque, most cells with lipid inclusions were localized in the median hyperplastic layer of the intima (10%). The muscular layer was characterized by the lowest content of cells with lipid inclusions both in the unaffected intima and atherosclerotic lesions (from 0.75% in unaffected intima to 5% plaques). Among the intimal smooth muscle cells of various shapes, the cells with lipid inclusions were most often found in the stellate cell subpopulation (5-35%). A possible role of stellate cells in atherogenesis is discussed.     

The content of lipoperoxidation products in normal and atherosclerotic human aorta.

To evaluate the role of lipid oxidation in atherogenesis the levels of lipid- and protein-bound products of peroxidation in normal and atherosclerotic areas of human aorta were investigated. The level of fluorescent (360/430 nm) lipid products was measured in chloroform-methanol extracts of aortic tissue. Normal intima, initial lesions and fatty streaks had a similar content of fluorescent substances. On the other hand, high level of fluorescent products was found in atherosclerotic plaques. Cholesterol covalently bound to proteins, which serve as a marker of lipoperoxidation, was measured by high performance liquid chromatography after mild alkaline hydrolysis of delipidated tissue protein samples. The levels of protein-bound cholesterol in initial lesions and fatty streaks were close to its content in uninvolved intima (59 +/- 18 and 92 +/- 18 vs. 70 +/- 13 nmol/g protein). The content of covalently bound cholesterol in atherosclerotic plaques was dramatically higher (90-fold) than in the normal tissue. In addition to protein-bound cholesterol, considerable amount of lipofuscin was revealed in the cells of atherosclerotic plaques, but not in the cells of normal intima, initial lesions or fatty streaks. Thus, the contents of all investigated lipid- and protein-bound products of lipoperoxidation in earlier atherosclerotic lesions were similar to their levels in normal tissue. It can be due to a low rate of oxidized product formation and/or high rate of its degradation in or elimination from the vessel wall.     

Cytotoxicity and transcriptional activation of stress genes in human liver carcinoma cells (HepG2) exposed to cadmium chloride

To evaluate the role of lipid oxidation in atherogenesis the levels of lipid- and protein-bound products of peroxidation in normal and atherosclerotic areas of human aorta were investigated. The level of fluorescent (360/430 nm) lipid products was measured in chloroform-methanol extracts of aortic tissue. Normal intima, initial lesions and fatty streaks had a similar content of fluorescent substances. On the other hand, high level of fluorescent products was found in atherosclerotic plaques. Cholesterol covalently bound to proteins, which serve as a marker of lipoperoxidation, was measured by high performance liquid chromatography after mild alkaline hydrolysis of delipidated tissue protein samples. The levels of protein-bound cholesterol in initial lesions and fatty streaks were close to its content in uninvolved intima (59 ± 18 and 92 ± 18 vs 70 ± 13 nmol/g protein). The content of covalently bound cholesterol in atherosclerotic plaques was dramatically higher (90-fold) than in the normal tissue. In addition to protein-bound cholesterol, considerable amount of lipofuscin was revealed in the cells of atherosclerotic plaques, but not in the cells of normal intima, initial lesions or fatty streaks. Thus, the contents of all investigated lipid- and protein-bound products of lipoperoxidation in earlier atherosclerotic lesions were similar to their levels in normal tissue. It can be due to a low rate of oxidized product formation and/or high rate of its degradation in or elimination from the vessel wall.     

3H-thymidine incorporation into human aorta cells in primary culture. An autoradiographic study

Primary cell culture has been obtained from intima and media of unaffected zones of human aorta and from atherosclerotic lesions (fatty infiltration, fatty streaks, atherosclerotic plaques). Cellular polymorphism was found in these cultures. Four morphological types of aortic cells are described: elongated, asymmetric, polygonal, and stellate cells. These cell types also differed in their proliferative activity. On the 7th day of culturing, polygonal cells do not incorporate 3H-thymidine; the thymidine index of other three cell types was similar. The thymidine index of medial cells was higher than that of intimal ones.     

Cyclic 3',5'-AMP phosphodiesterase of rabbit aorta.

Cyclic AMP and cyclic GMP phosphodiesterase activities (3' : 5'-cyclic AMP 5'-nucleotidohydrolase, EC 3.1.4.17) were demonstrated in the isolated intima, media, and adventitia of rabbit aorta. The activity for cyclic AMP hydrolysis in the intima was 2.7-fold higher than that for cyclic GMP hydrolysis. The activity for cyclic AMP hydrolysis in the media was approximately equal to that for cyclic GMP hydrolysis, but in the adventitia, cyclic GMP hydrolytic activity was 2.1-fold higher than cyclic AMP hydrolytic activity. Distribution of the activator of the phosphodiesterase was studied in the three layers. Each layer contained the activator. The activator was predominantly localized in the smooth muscle layer (the media). The effect of the activator and Ca2+ on the media cyclic AMP and cyclic GMP phosphodiesterase was also briefly studied. The activity of the cyclic GMP phosphodiesterase was stimulated by micromolar concentration of Ca2+ in the presence of the activator. However, the activity of the cyclic AMP phosphodiesterase was not significantly stimulated by Ca2+ up to 100 muM in the presence of the activator. Above 90% of cyclic nucleotide phosphodiesterase activity in the whole aorta was found to be derived from the media. A major portion (60-70%) of the media enzyme was found in 105 000 times g supernatant. Cyclic AMP phosphodiesterase in the supernatant was partially purified through Sepharose 6B column chromatography and partially separated from cyclic GMP phosphodiesterase. Using a partially purified preparation from the 105 000 times g supernatant the main kinetic parameters were specified as follows: 1) The pH optimum was found to be about 9.0 using Tris-maleate buffer. The maximum stimulation of the enzyme by Mg2+ was achieved at 4mM of MgC12. 2) High concentration of cyclic GMP (0.1 mM) inhibited noncompetitively the enzyme activity, and the activity was not stimulated at any tested concentration of cyclic GMP. 3) Activity-substrate concentration relationship revealed a high affinity (Km equals 1.0 muM) and low affinity (Km equals 45 muM) for cyclic AMP. The homogenate and 105 000 times g supernatant of the media also showed non-linear kinetics similar to the Sepharose 6B preparation and their apparent Km values for cyclic AMP hydrolysis were 1.2 muM and 36-40 muM and an enzyme extracted by sonication from 105 000 times g precipitate also exhibited non-linear kinetics (Km equals 5.1 muM and 70 muM). 4) Papaverine exhibited much stronger inhibition on the aorta cyclic AMP phosphodiesterase (50% inhibition of the intima enzyme, I5 o at 0.62 muM, I5 o of the media at 0.62 muM and I5 o of the adventitia at 1.0 muM) than on the brain (I5 o at 8.5 muM) and serum (I5 o at 20 muM) cyclic AMP phosphodiesterase, while theophylline inhibited these enzymes similarly. However, cyclic GMP phosphodiesterases in all tissues examined were inhibited similarly, not only by theophylline but also by papaverine.     

Regulation of cyclic GMP, cyclic AMP and lactate dehydrogenase by putative neurotransmitters in the C6 rat glioma cell line.

In C6 cells norepinephrine and dopamine caused transient increases in cyclic GMP and cyclic AMP, as well as an induction of lactate dehydrogenase. All of these responses were blocked by l-propranolol, suggesting mediation by a β-receptor. Phentolamine potentiated the NE-increased cAMP levels by 5-fold when NE was used at suboptimal doses, suggesting the presence of α-adrenergic receptors in C6 cells. Carbamylcholine decreased the levels of both cyclic nucleotides, with hexamethonium partially reversing the effect on cyclic GMP. Dibutyryl-cyclic GMP or carbamylcholine reduced catecholamine-induced cyclic AMP levels. Serotonin increased cyclic GMP levels 60% and decreased cyclic AMP levels 36%. Calcium- and magnesium-free media inhibited the norepinephrine-induced levels of cyclic GMP and cyclic AMP respectively.     

Numbers of cells and cell proliferation in intima of different human arteries

Increased cell proliferation in early atherosclerotic lesions is recognized as an essential event of atherogenesis but the levels of cell proliferation in different stages of atherosclerotic plague formation in different types of human large arteries are still insufficiently studied. In the present work, we studied intima thickness and proliferation of newly "infiltrates" hematogenous and resident cells in atherosclerotic lesions of the carotid and coronary arteries and compared these parameters with those in the aorta, reported by us in earlier publication. Analysis of intima thickness and proliferation in grossly unaffected intima and in different types pf atherosclerotic lesions (initial lesions, fatty streaks, lipofibrous, plaques, and fibrous plaque) revealed that although there were similar tendencies in the change of the infiltration levels of hematogenous cells and proliferation in different types of arteries, there were significant quantitative differences between different types of arteries. Hematogenous cells in lipofibrous plaques of the coronary and carotid arteries were found to account for a third and almost for a half of the total cell population, respectively, while atherosclerotic lesions in the aorta, as it has been shown by us earlier, to contain no more than 15% ofhematogenous cells. This suggests that the contribution of hematogenous cells to the development of atherosclerosis in the carotid and the coronary artery appears to be more significant than that in the aorta. Despite the differences in numbers of accumulating hematogenous cells in the intima, a similar "bell-shaped" dependence of cell numbers on the lesion type, involved in the following sequence: unaffected intima-initial lesions-fatty streaks-lipofibrous plaques-fibrous plaques, was detected in the coronary and carotid arteries. The visualization of proliferating cells (PCNA-positive) in atherosclerotic and unaffected zones of the coronary and carotid arteries revealed similar patterns. The maximum numbers of PCNA-positive resident cells were identified in lipofibrous plaques. The changes in the total cell numbers were accompanied by the changes in the numbers of both proliferating resident cells and proliferating hematogenous cells.     

Changes in rat adrenal cyclic nucleotides during normal and neoplastic growth

In an attempt to correlate changes in cyclic nucleotide levels with in vivo growth of the rat adrenal gland we have measured adrenal cyclic AMP and cyclic GMP in normal, hyperplastic, and neoplastic rat adrenals. The first group of animals were subject to either unilateral adrenalectomy (ADX) or acute hypophysectomy 1 h prior to unilateral adrenalectomy (HADX). Cyclic nucleotides were measured in the contralateral adrenal post-operatively. In HADX rats cyclic GMP rose steadily throughout the 7 day study period, while ADX rats exhibited significant decreases in adrenal cyclic GMP. Cyclic AMP remained approximately 1.5 pm/mg tissue in HADX rats, while in ADX rats there was significant elevation of adrenal cyclic AMP at all time points. Cyclic GMP/cyclic AMP ratios remained constant in HADX animals; however, the growing adrenals of ADX animals exhibited depressed cyclic GMP/cyclic AMP ratios at all time periods.Adrenal hyperplasia was induced in a seond group of animals by a transplantable, corticotropin-secreting, pituitary tumor. Adrenals from age-matched animals served as controls. Adrenal cyclic AMP was significantly elevated in tumor-bearers at a time correspinding to the peak accumulation of adrenal weight, protein and DNA in these animals. In contrast, adrenal cyclic GMP in both tumor-beares and control animals fell steadily throughout the study period. Cyclic GMP/cyclic AMP ratios of control animals decreased from 2 to 3 weeks post-transplant remaining at the 3 week value during the period corresponding to rapid adrenal growth in tumor-bearers. The cyclic GMP/cyclic AMP ratio in the hyperplastic adrenals of tumor-bearers decreased steadily throughout their rapid growth period, suggesting a positive correlation between adrenal growth and depression of the cyclic GMP/cyclic AMP ratio.Cyclic nucleotide levels in neoplastic adrenals of rats bearing the transplantable adrenocortical carcinoma 494 were compared with cyclic nucleotides in normal rat adrenal glands. Cyclic AMP was not different in the two groups. However, the cyclic GMP content of neoplastic adrenals was significantly lower than that of normal adrenal tissue, causing a suppression of the cyclic GMP/cyclic AMP ratio in the neoplastic tissue. Thus, measurement of adrenal cyclic nucleotides in both hyperplastic and neoplastic rat adrenal glands suggests that adrenal growth in vivo may be characterized by a depression of the cyclic GMP/cyclic AMP ratio.     

Preparation of cyclic nucleotide antisera with thyroglobulin-cyclic nucleotide conjugates

Antisera to cyclic AMP and cyclic GMP were obtained by immunizing rabbits with antigens prepared by conjugating the 2'0-succinyl derivative of the cyclic nucleotides to thyroglobulin. The cyclic nucleotide-thyroglobulin conjugates were injected intradermally into multiple sites on the backs of the animals. This immunization procedure resulted in the production of antiserum in four of five animals capable of binding at a final serum dilution of greater than 1:10,000, 20% of the corresponding iodinated cyclic nucleotide derivative added. The antisera were also highly specific. The antiserum for cyclic AMP had a 2500-fold or greater relative affinity for cyclic AMP than other nucleotides or nucleosides, while that for cyclic GMP had a 5000-fold or greater affinity for 2'0 acetylated nucleotides or nucleosides except for acetylated cyclic IMP. The obstacles to measuring cyclic nucleotides, particularly cyclic GMP, in tissues were also overcome by refining and simplifying the methods for iodination, purification and assay. Furthermore, a "disequilibrium" incubation was developed as an alternative to the acetylation method to increase the sensitivity of the radioimmunoassay. Thus, the levels of both cyclic GMP and cyclic AMP can be determined rapidly and easily in the same tissue sample.     

Amylase secretion by rabbit parotid gland. Role of cyclic AMP and cyclic GMP.

Amylase secretion and changes in the levels of cyclic AMP and GMP were studied in rabbit parotid gland slices incubated in vitro with a variety of neurohumoral transmitters, their analogs and inhibitors. Cyclic GMP levels increased 8-fold 5 min after exposure to carbachol (10(-4) M), without a change in cyclic AMP levels; amylase output also rose. These effects were completely inhibited by muscarinic blockade with atropine, but were unaffected by alpha-adrenergic blockade with phenoxybenzamine. Epinephrine (4 - 10(-5) M) produced a rapid increase in the levels of both cyclic nucleotides and in amylase release. The increase in cyclic GMP level was inhibited by previous exposure of the slices to phenoxybenzamine, while the cyclic AMP rise was prevented by the beta-blocking agent, propranolol. Pure alpha-adrenergic stimulation with methoxamine (4 - 10(-4) M) produced modest elevations in cyclic GMP content and amylase output, effects blocked by pre-treatment of slices with either atropine or phenoxybenzamine. At a concentration of 4 - 10(-6) M, isoproterenol (a beta-agonist) failed to affect cyclic GMP levels, but promptly stimulated increases in cyclic AMP levels, and after a short lag, amylase secretion. At a higher dose (4 - 10(-5) M) isoproterenol produced elevations in the levels of both nucleotides. The carbachol-induced effects on cyclic GMP content and amylase release were greatly potentiated by the addition of isoproterenol (4 - 10(-6) M). These data strongly suggest that cholinergic muscarinic agonists and alpha-adrenergic agonists stimulate amylase output in rabit parotid gland by mechanisms involving cyclic GMP. The atropine-sensitive intracellular events effected by alpha-stimulation may be dependent upon endogenous generation of acetylcholine. Both cyclic nucleotides seem to be required for the early rapid secretion of amylase. The unique responses achieved by the combination of carbachol and isoproterenol suggest that isoproterenol may increase the sensitivity of this tissue to the effects of cholinergic stimuli.     

Effects of sera types and cell density on the concentration of cyclic nucleotides in normal and simian virus transformed mouse fibroblasts

The effects of serum and cell density on the concentration of cyclic AMP, cyclic GMP in normal mouse fibroblasts cells (3T3 cells) and their Simian Virus 40 transformed derivative (SV3T3 cells) were studied. 3T3 cells grown in 10% foetal bovine serum exhibit density dependent inhibition of growth and associated with this in an increase in the concentration of cyclic AMP, a decrease in the concentration of cyclic GMP and an increase in the ratio (cyclic AMP/cyclic GMP) of the cyclic nucleotides. 3T3 cells grown in 10% newborn calf serum exhibit a higher saturation density and this is associated with a low concentration of cyclic AMP and a high concentration of cyclic GMP. SV3T3 cells grown in either 10% foetal bovine serum or 10% newborn calf serum show high saturation densities and this is associated with a low and decreasing concentration of cyclic AMP and a high concentration of cyclic GMP. When the level of the cyclic AMP in both cell lines was artificially raised by adding dibutyryl cyclic AMP and theophylline to the growth media, the cells grew to low densities.     

Comparison of cyclic nucleotide specificity of guanosine 3',5'-monophosphate-dependent protein kinase and adenosine 3',5'-monophosphate-dependent protein kinase.

Guanosine 3',5'-monophosphate-dependent protein kinase (cyclic GMP-dependent protein kinase) and adenosine 3',5'-monophosphate-dependent protein kinase (cyclic AMP-dependent protein kinase) exhibited a high degree of cyclic nucleotide specificity when hormone-sensitive triacylglycerol lipase, phosphorylase kinase, and cardiac troponin were used as substrates. The concentration of cyclic GMP required to activate half-maximally cyclic dependent protein kinase was 1000- to 100-fold less than that of cyclic AMP with these substrates. The opposite was true with cyclic AMP-dependent protein kinase where 1000- to 100-fold less cyclic AMP than cyclic GMP was required for half-maximal enzyme activation. This contrasts with the lower degree of cyclic nucleotide specificity of cyclic GMP-dependent protein kinase of 25-fold when histone H2b was used as a substrate for phosphorylation. Cyclic IMP resembled cyclic AMP in effectiveness in stimulating cyclic GMP-dependent protein kinase but was intermediate between cyclic AMP and cyclic GMP in stimulating cyclic AMP-dependent protein kinase. The effect of cyclic IMP on cyclic GMP-dependent protein kinase was confirmed in studies of autophosphorylation of cyclic GMP-dependent protein kinase where both cyclic AMP and cyclic IMP enhanced autophosphorylation. The high degree of cyclic nucleotide specificity observed suggests that cyclic AMP and cyclic GMP activate only their specific kinase and that crossover to the opposite kinase is unlikely to occur at reported cellular concentrations of cyclic nucleotides.     

Cyclic nucleotide phosphodiesterases of Hyalophora cecropia silkmoth fat body.

Two soluble forms of 3':5'-cyclic-nucleotide phosphodiesterase (o':5'-cyclic-nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) were found in the larval fat body of the silkmoth Hyalophora cecropia. These differ in elution profile on Sephadex G-200, solubility in ammonium sulfate, metal ion requirements and kinetic properties. Phosphodiesterase I has Km values of 11 muM and 1.8 muM for cyclic AMP and cyclic GMP, respectively, has 5-fold greater maximal activity with cyclic AMP than with cyclic GMP, and is activated by Mg2+ and Co2+, and inhibited by EDTA. phosphodiesterase II has Km values of 625 muM and 125 muM for cyclic AMP and cyclic GMP, respectively, has similar maximal activity with both substrates, and is not activated by divalent metal ions or inhibited by EDTA. Cyclic nucleotides and methylxanthines competitively inhibit both enzymes. Phosphodiesterase is found in both soluble and particulate fractions of homogenates. Total activity is highest during the larval stage of the insect, drops markedly following pupation, and rises again during pharate adult development.     

Phosphodiesterase Activities for Cyclic Nucleotides in Nerve Endings from the Bovine Posterior Pituitary Gland

Abstract: The cyclic nucleotide phosphodiesterase (PDE) activities were studied in a nerve ending fraction from bovine neural lobes. Most of the activity was particulate and unaffected by calcium. Lineweaver-Burk plots for this fraction showed negative cooperativity with apparent K _m values for cyclic AMP of 11 μ M and for cyclic GMP of 4 μ M . The soluble activities for both cyclic nucleotides were activated by calcium and inhibited by calmodulin-binding drugs (trifluoperazine and calmidazolium). The apparent K _m values were 50 μ M for cyclic AMP and 20 μ M for cyclic GMP for the soluble activities. Sucrose density gradients resolved the soluble activities into two peaks. The activity with the higher sedimentation rate (MW 122,000 daltons) hydrolysed both cyclic nucleotides and was calcium-calmodulin-dependent. The other peak (MW 47,000 daltons) had a higher affinity for cyclic AMP than for cyclic GMP and was calcium-independent. Solubilized particulate activities gave two main peaks on the density gradient, both calcium-independent. One was mainly for cyclic AMP (MW 47,000 daltons) and the other mainly for cyclic GMP (MW 133,000 daltons). The function of PDEs in relation to secretion was discussed.     

Cyclic nucleotide phosphodiesterase activities of pig coronary arteries.

Most (85% or more) of the cyclic nucleotide phosphodiesterase (3' :5' -cyclic-AMP 5'-nucleotidohydrolase, EC 3.1.4.17) activity of pig coronary arteries was found in the 40 000 times g supernatant fraction of homogenates of the intima plus media layer. Chromatography of the soluble fraction of this layer on DEAE-cellulose resolved two phosphodiesterase activities and a heat stable, non-dializable activator. Peak I activity had apparent Km values of 2-4 muM for cyclic GMP and 40-100 muM for cyclic AMP. Peak II activity was relatively specific for cyclic AMP and exhibited apparent negatively cooperative behavior. Peak I but not peak II activity could be stimulated 3-8-fold by the addition of the boiled activator fraction or a boiled crude supernatant fraction. Cyclic AMP hydrolysis by peak I or peak II was more rapid in the presence of Mn-2+ than Mg-2+, but the latter promoted hydrolysis of cyclic GMP by peak I more effectively than did Mn-2+ in the presence of activator. In the absence of added metals, ethylene bis(oxyethylenenitriol)tetra-acetic acid (EGTA) and EDTA both inhibited hydrolysis of cyclic AMP and cyclic GMP by phosphodiesterase activities in the supernatant fraction and in peak I, but EDTA produced more complete inhibition at lower concentrations than did EGTA. Imidazole (1 muM to 10 mM) had virtually no effect on the hydrolysis of cyclic AMP or cyclic GMP catalyzed by either of the two separated peaks or by total phosphodiesterase activities in crude supernatant or particulate fractions.     

A possible involvement of cya gene in the synthesis of cyclic guanosine 3':5'-monophosphate in E. coli.

Based on the following genetical experiments, the cya gene in E. coli was shown to be involved in the synthesis of both cyclic AMP and cyclic GMP. First, all five independent cya-deficient mutants accumulated exceedingly low amounts of cyclic GMP. Second, the ability to form both cyclic AMP and cyclic GMP was simultaneously restored by transduction of an intact cya locus to one of the above cya-deficient mutants. Third, a spontaneous revertant from one of the above mutants regained the synthetic activity for cyclic GMP as well as for cyclic AMP. Fourth, the characteristic of a strain overproducing cyclic GMP was co-transduced with the cya locus. These results suggest that the synthesis of both cyclic GMP and cyclic AMP is mediated by the same enzyme, adenylate cyclase, Interestingly, a reciprocal effect of glucose starvation was observed on the accumulation of both cyclic nucleotides. The formation of cyclic AMP was greatly enhanced on glucose starvation, whereas that of cyclic GMP proceeded at a slower rate than in the presence of glucose. This effect was observed only in cells carrying normal cya and crp genes, but not in a cya-altered or a crp-deficient strain.     

Activation of murine lymphocytes by cyclic guanosine 3′,5′-monophosphate: Specificity and role in mitogen action

Cyclic guanosine 3′,5′-monophosphate (cyclic GMP) stimulates nucleic acid synthesis in lymphocytes, and has been implicated as the intracellular effector of the actions of mitogenic agents on these cells. In the present study, we examined the specificity of the mitogenic activity of cyclic GMP and of its 8-bromo (Br) derivatives, and the effects of the T cell mitogens, concanavalin A, phytohemagglutinin, and staphylococcal entertoxin B (SEB) on the cyclic GMP content and guanylate cyclase activity of mouse splenic lymphocytes. Cyclic GMP and guanosine modestly increased the incorporation of [³H]thymidine into DNA by cultured lymphocytes, but were far less effective than their 8-Br-derivatives. However, on a molar basis the mitogenic activity of both 8-Br-guanosine and 8-Br-5′-GMP exceeded that of 8-Br-cyclic GMP, when tested in the presence and absence of serum in the culture media. Combined addition of maximal doses of these nucleotides did not give additive stimulatory effects, suggesting an action on a common subpopulation of cells, and possibly a common mechanism. By contrast, cyclic AMP, 8-Br-cyclic AMP, 8-Br-adenosine, cholera toxin and prostaglandin E₁ suppressed both basal [³]thymidine incorporation and stimulation of this parameter by T-cell line mitogens and the guanosine nucleotides. Rapid effects of concanavalin A, phytohemagglutinin, SEB, guanosine, 5′-GMP, 8-Br-guanosine, and 8-Br-5′-GMP on the cyclic GMP content of murine lymphocytes could not be demonstrated. Similarly, concanalin A, phytohemagglutinin and SEB failed to alter guanylate cyclase activity when added directly to cellular homogenates or pre-incubated with intact cels. Conversely, carbamylcholine rapidly increased lymphocyte cyclic GMP but was not mitogenic.These results are consistent with the hypothesis that cyclic GMP and cyclic AMP are antagonistic in their influence on lymphocyte mitogenesis. However, they also demonstrate that related nucleotides are more potent mitogens than cyclic GMP and suggest that activation of murine lymphocytes by concanavalin A, phytohemagglutinin and SEB may not be mediated by rapid increases in cellular cyclic GMP content. Since high concentrations of exogenous cyclic GMP and related nucleotides must be used to influence DNA synthesis, the biologic significance of this effect remains uncertain.     

Effect of diapause hormone on cyclic nucleotide metabolism in developing ovaries of the silkworm, Bombyx mori

1. The content of cyclic AMP and cyclic GMP in ovaries of the silkworm, Bombyx mori, changed differently depending on development. 2. The extirpation of suboesophageal ganglion caused cyclic AMP to decrease and cyclic GMP to increase at different developmental stages. 3. The reversed changes in the cyclic nucleotides were brought about by implantation of the ganglion or by injection of the diapause hormone preparation. 4. No change in both cyclic nucleotides was induced in ovaries during the first 24 hr after an injection of the hormone, but the clear effects appeared after a lag phase of a few days.     

Comparison of cyclic nucleotide specificity of guanosine 3′,5′-monophosphate-dependent protein kinase and adenosine 3′,5′-monophosphate-dependent protein kinase

Guanosine 3′,5′-monophosphate-dependent protein kinase (cyclic GMP-dependent protein kinase) and adenosine 3′,5′-monophosphate-dependent protein kinase (cyclic AMP-dependent protein kinase) exhibited a high degree of cyclic nucleotide specificity when hormone-sensitive triacylglycerol lipase, phosphorylase kinase, and cardiac troponin were used as substrates. The concentration of cyclic GMP required to activate half-maximally cyclic dependent protein kinase was 1000- to 100-folds less than that of cylic AMP with these substrates. The opposite was true with cyclic AMP-dependent protein kinase where 1000- to 100-fold less cyclic GMP was required for half-maximal enzyme activation. This contrasts with the lower degree of cyclic nucleotide specificity of cyclic GMP-dependent protein kinase of 25-fold when histone H2b was used as a substrate for phosphorylation. Cyclic IMP resembled cyclic AMP in effectiveness in stimulating cyclic GMP-dependent protein kinase but was intermediate between cyclic AMP and cyclic GMP in stimulating cyclic. AMP-dependent protein kinase. The effect of cyclic IMP on cyclic GMP-dependent protein kinase was confirmed in studies of autophosphorylation of cyclic GMP-dependent protein kinase where both cyclic AMP and cyclic IMP enhanced autophophorylation. The high degree of cyclic nucleotide specificity observed suggests that cyclic AMP and cyclic GMP activate only their specific kinase and that crossover to the opposite kinase is unlikely to occur at reported cellular concentrations of cyclic nucleotides.