Effects of radioprotectors on the cAMP and cGMP systems

Summary The sulphur-containing radioprotectors mercaptoethylamine (MEA), aminoethylisothiourea (AET), 2-aminothiazoline, 4-oxo-2-aminothiazoline, and S-S-3-oxapentane-1,5-diisothiourea, and the radioprotective biogenic amines serotonin, histamine, and dopamine, caused the elevation of cAMP content and intensified the rate of cAMP-dependent protein phosphorylation in tissues of animals following intraperitoneal injection at radioprotective doses. Biogenic amines stimulated the adenylate cyclase activity in membrane preparations from liver, spleen, and small-intestine mucosa; sulphur-containing radioprotectors caused no such effects. None of the radioprotectors affected cAMP and cGMP phosphodiesterases in vitro. AET and MEA inhibited guanylate cyclase in vitro, whereas serotonin and dopamine stimulated the enzyme. A biphasic change in the level of cGMP was observed in tissues after the administration of MEA and AET (more than 2-fold fall by 1–3 min after the administration of drug and 1.4-fold rise after 15–20 min); serotonin and dopamine caused a slow rise in the cGMP level; the cAMP/cGMP ratio in liver showed biphasic changes in level during the 20 min following injection of serotonin.The data obtained support the conclusion that the action of radioprotectors on cellular metabolism in animals may be mediated by the cAMP system. The reciprocal regulation of radioresistance by cAMP and cGMP is unlikely to exist.     

The role of calcium in regulation of cyclic nucleotide content in human umbilical artery.

In term gestational human umbilical artery segments incubated in room air at 37 degrees, histamine, acetylcholine, bradykinin, K+, and serotonin (agonists that cause contraction) cause accumulation of guanosine 3':5'-monophosphate (cGMP) without altering the content of adenosine 3':5'-monosphophate (cAMP); prostaglandin E1 (PGE1), which relaxes the artery, causes cAMP accumulation without affecting the cGMP content (Clyman, R. I., Sandler, J.A., Manganiello, V.C., and Vaughan, M. (1975) J. Clin. Invest., in press). It has been postulated that Ca-2+ is important in the regulation of cyclic nucleotides in other tissues. In the umbilical artery the control of cAMP content by PGE1 was independent of Ca-2+. After incubation in Ca-2+-free medium, the c GMP contentof the artery segments was decreased by 50% and was unaffected by histamine, acetylcholine, bradykinin, and K+. Readdition of Ca-2+ (2.7 mM) or Sr-2+ (3.6 mM) to the medium partially restored the basal cGMP content and the agonist effects on the cGMP content. However, Sr-2+ was not as effective as Ca-2+ in this regard. Ionophores A23187 and X537A (agents that facilitate Ca-2+ movement through membranes) mimicked the effects of these Ca-2+-dependent agonists on cGMP content. Incubation with the phosphodiesterase inhibitor 3-isobutyl-1-methyl xanthine (0.1 mM) increased both the basal content of cGMP and the histamine-induced accumulation 3-fold. This effect was dependent on the presence of Ca-2+ also. Accumulation of cGMP induced by serotonin, on the other hand, was not diminished in Ca-2+-depleted arteries and, in fact, seemed to be inhibited by 2.7 mM Ca-2+. These observations are consistent with the existence in the umbilical artery of two separate mechanisms for control of cGMP synthesis that are influenced differently by Ca-2+.     

Dynamics of the change in cyclic nucleotide content in wound tissues]

Changes in the content of cyclic nucleotides (cAMP and cGMP) in the wound tissues (muscles and granulations) were studied in experiments on rats. A wound with skin defect in the dorsal area with a crushed underlying muscle served as an experimental model. It was shown that the cAMP content in the muscular tissue rises twice: during the 1st day and to a greater measure on the 7th day. The cGMP content slightly increases on the 1-4th day, drops on the 7th day and increases cAMP concentration changes similarly to that inthe muscular tissue: it increases on the 7th day and drops on the 14th day. On the contrary, the cGMP content curve in granulations is more monotonous, only a slight increase being observed on the 7th day.     

Changes in cyclic nucleotide levels during embryonic development of chick hearts

The effects of isoproterenol, acetylcholine (Ach), and adenosine, on cyclic AMP (cAMP) and cyclic GMP (cGMP) contents were examined in chick hearts at various stages of embryonic development. The basal cAMP content was highest (87.7 +/- 1.3 pmol/mg protein) in young (3-day) embryonic chick hearts and decreased during development (9.6 +/- 0.6 pmol/mg protein in 9-19-day-old hearts). On the other hand, the cGMP content was lowest (45.5 +/- 2.3 fmol/mg protein) in young (3-day) embryonic chick hearts and increased during development (338 +/- 15.0 fmol/mg protein in 14-19-day-old hearts). Iso increased the cAMP concentration in embryonic hearts at all ages. Ach and Ado had no effect on the cAMP content at all ages. However, the Isoproterenol-induced stimulation of cAMP was inhibited by Ach and Adenosine at all ages. In young embryonic hearts, Ach and Ado increased cGMP concentration only slightly, whereas these agents caused a substantial increase in cGMP concentration in the older hearts. Thus, there was a clear age difference in the effects of Ach and Adenosine on the cGMP and cAMP concentrations. Nitroprusside and hydrogen peroxide increased cGMP concentration in older hearts (greater than 5-day-old) but not in the 3-day-old embryonic hearts. Thus, guanylate cyclase activity may be low in young (3-day-old) hearts. It summary, the cGMP level is very low in young embryonic chick hearts, and increases markedly during development. The changes in cGMP are reciprocal to those of cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyclic nucleotide metabolism in mouse epididymal spermatozoa during capacitation in vitro

The role of cyclic nucleotides in sperm capacitation is equivocal. Using conditions known to support mouse sperm capacitation after 120 min incubation in vitro, the cAMP and cGMP contents of epididymal spermatozoa were measured and the cGMP/cAMP ratio determined. The initial high cAMP content detected upon release of spermatozoa decreased within 30 min to a lower plateau, which was then maintained throughout incubation. With the cGMP content remaining approximately constant, the cGMP/cAMP ratio increased over 120 min. In the presence of 2 mM caffeine, an increased cAMP content was noted at 0 and 30 min before a fall to the plateau level. To investigate cyclic nucleotide metabolism, adenylate cyclase and phosphodiesterase activities were compared in two sperm populations, one essentially uncapacitated and the other incubated for 120 min. Adenylate cyclase activity, higher in the presence of 2 mM Mn²⁺ compared to Mg²⁺, showed increased activity at 120 min compared to 30 min incubation, while phosphodiesterase activity decreased during this period. The ability of spermatozoa to form adenosine and inosine from cAMP indicated endogenous 5′-nucleotidase and deaminase, as well as phosphodiesterase, activities. Although the endogenous cAMP content appeared to remain constant during the time that acrosome loss, hyperactivated motility and fertilizing ability can be demonstrated, activities of the enzymes responsible for cAMP metabolism indicate an increased potential for cAMP availability and turnover. The increased cGMP/cAMP ratio may also play a role during capacitation.     

Cyclic nucleotide metabolism in the human erythrocyte

Erythrocytes, which show little or no guanylate or adenylate cyclase activity, take up cyclic nucleotides from blood. Studies were done by incubating human erythrocytes in isotonic medium with the dibutyryl derivatives of cAMP and cGMP and in a hypotonic medium in which the cells are partially hemolyzed and, therefore, freely permeable to cAMP and cGMP. At cAMP and cGMP concentrations of 50 microM and above, the amount of 14CO2 generated from 1-14C-glucose was decreased significantly. This effect was suppressed by 4.6 mM theophylline. Inosine and ribose, which are catabolites of cAMP and cGMP also decreased formation of 14CO2 from 1-14C-glucose. Accordingly, it is postulated that in the absence of theophylline, the activity of phosphodiesterase resulted in AMP and GMP formation. These mononucleotides enter into the purine salvage pathways to form ribose phosphate. Additionally, the production of lactate and 2,3-diphosphoglycerate (2,3-DPG) was measured in human erythrocytes after incubation with the dibutyryl derivatives of cAMP (bt2-cAMP) and cGMP (bt2-cGMP). At a concentration of 0.1 microM, bta2-cGMP inhibits lactate production at 60 min (p less than 0.01). Slight increases in 2,3-DPG were not statistically significant. Catabolism of cyclic nucleotides may prevent diffusion equilibria allowing for the erythrocyte's continuous uptake of cyclic nucleotides and providing a detoxification mechanism that could compensate for conditions in which elevations of these substances are observed.     

Effect of the H1-histamine-like agonist material extracted from bovine spleen on 3': 5'-cyclic nucleotides content in guinea-pig ileal smooth muscle

The effect of a water-soluble "histamine-free" splenic material that mimics the H1-receptor mediated contractile action of histamine on the guinea-pig ileum has been studied upon concentrations of cGMP and cAMP in slices of this smooth muscle preparation. The splenic extract induced a rapid and sustained decrease in the concentration of cGMP accompanied by a slow decrease in cAMP content in the ileal tissue. These results indicate that the smooth muscle-stimulating agent in splenic extract had no increasing effect on cGMP content as could be expected from the hypothesis that cGMP and cAMP might mediate the smooth muscle contraction and relaxation respectively. The data are not compatible with the general hypothesis that the action of histamine and histamine-like agonists on H1-receptors is associated with an increased concentration of cGMP.     

Cyclic nucleotides in the ontogeny of the chick thymus

The dynamics of cAMP and cGMP content of the thymus homogenate from developing chick embryos and chickens was studied during ante- and postnatal development. Changes in the content of cyclic nucleotides bear an oscillatory pattern. At the early stages of embryogenesis (9 to 11 days of incubation) the content of cyclic nucleotides was low and gradually increased by 13 days of incubation. As the development proceeded, the quantitative and qualitative rearrangement of the thymus cellular composition reflected in changes in the content of cyclic nucleotides. At the same time the curves of cyclic nucleotide content became antiphasic. These reciprocal cAMP to cGMP ratios might reflect the cyclic and synchronous reproduction and functional development of the main bulk of the thymus cellular elements. The maximum content of cAMP and the minimum content of cGMP were recorded on the 17th day of embryogenesis.     

Biochemical changes in progressive muscular dystrophy. XI. Cyclic nucleotides in the skeletal and cardiac muscle of normal and dystrophic mice

cAMP and cGMP contents were determined in the skeletal and cardiac muscle of normal and dystrophic mice. cAMP content increased in the dystrophic muscle at every stage of the disease whereas cGMP content decreased in the preliminary stages and increased at the terminal stage of the disease. The content of both nucleotides per heart was not affected in murine dystrophy. Thus, levels of cyclic nucleotides appear to be selectively altered in dystrophic skeletal muscle.     

Cyclic nucleotides of human and animal glial tumors

Studies on the level of cyclic nucleotides (cAMP and cGMP) in human and animal glial tumours showed that the content of both nucleotides, especially that of cAMP, decreases in all the tumours. The cAMP/cGMP ratio also drops down. Concurrently it appears to be the most consistent parameter of nucleotide metabolism both in brain tissue and in human or animal glial tumours. The growing tumour affects cAMP and cGMP metabolism not only in the involved but also in the other hemisphere. No principal differences between human and animal tumours have been revealed in the content of cyclic nucleotides and its variation in tumour tissue.     

cGMP-activated phosphodiesterase from human brain: kinetic and regulatory properties]

The kinetic and regulatory properties of cGMP-activated phosphodiesterase (PDE) from human brain were studied. In double reciprocal plots the enzyme activity is characterized by a linear dependence of cAMP and a nonlinear one for cGMP. Micromolar concentrations of cGMP accelerate cAMP hydrolysis (7-14-fold) with Ka for cGMP of 0.36 microM. Stimulation of cAMP hydrolysis is accompanied by a decrease of Km with no changes in Vmax. With a rise in the cGMP concentration above 5 microM PDE activation is changed by its inhibition. Both substrates act as competitive inhibitors towards each other. The Ki value for both cGMP and cAMP is 30 microM. After the increase in the cAMP (Bt)2 concentration the activation of 5 microM cAMP hydrolysis is accompanied by the enzyme inhibition. Both analogs competitively inhibit cGMP hydrolysis with Ki of 10 and 1500 microM for cGMP(Bt)2 and cAMP(Bt)2, respectively. The data obtained point to the existence of two binding sites for cyclic nucleotides, namely, a regulatory site which is highly specific for cGMP and a catalytic site responsible for the hydrolysis of the both substrates which displays no apparent specificity either for cAMP or for cGMP. The different affinity of natural and synthetic cyclic nucleotides for these sites is determined, to a large extent, by the amino groups in the 2nd and 6th positions of the purine ring.     

Changes in the cyclic nucleotides of rat thyroid, pituitary and plasma caused by methylthiouracil treatment.

Changes in the content of cyclic nucleotides (cAMP and cGMP) and related enzyme activities were observed in the rat thyroid, pituitary and plasma during the prolonged increase of endogenous TSH produced by treatment with methylthiouracil (MTU). Experiments were performed after 4 weeks treatment with MTU. The wet weight and cAMP content per wet weight of the thyroid increased 3 and 1.4 times respectively, but cGMP showed a slight decrease. Pituitary weight increased 1.3 times, but cAMP and cGMP content did not change. The cAMP level in plasma also increased about 1.3 times, but cGMP did not increase. The cAMP-phosphodiesterase activity in the thyroid, pituitary and plasma was increased 1.9, 1.4 and 1.3 times respectively after MTU treatment, while cGMP-phosphodiesterase showed no significant change. ATPase activity in the thyroid and pituitary was also increased more than 1.5 times after MTU treatment, while 5'-nucleotidase activitity decreased remarkably. These data indicate that the metabolism of the cyclic nucleotide system in the thyroid is stimulated by TSH.     

Effects of cyclic nucleotides on lipid biosynthesis in mouse mammary gland explants

The effects of dibutyryl cAMP, 1-methyl-3-isobutyl xanthine (MIX), cGMP, dibutyryl cGMP, and 8-bromo cGMP on the rate of lipid synthesis in mouse mammary gland explants were studied. Dibutyryl cAMP at 10(-4) M selectively inhibited the effect of prolactin on the rate of [14C]acetate incorporation into lipids. At 10(-3) M, dB-cAMP inhibited the effects of insulin, insulin plus cortisol, and prolactin. The phosphodiesterase inhibitor, MIX, inhibited both basal and prolactin-stimulated incorporation rates in a concentration-dependent fashion. These data suggest an inhibitory role for cAMP in the regulation of lipogenesis in the mammary gland. Cyclic GMP, db-cGMP, and 8-bromo cGMP were all without effect on either basal or prolactin-stimulated incorporation rates. Therefore, it appears that cGMP, by itself, is not involved in the regulation of lipogenesis in the mammary gland.     

Decrease in cAMP levels and increase in cGMP levels in the small intestine mucosa after treatment with fluoride

Sodium fluoride was studied for its effect on the cAMP and cGMP amount in the small intestine mucosa of rats under conditions of acute and chronic experiments. A significant decrease in the cAMP level and an increase in the cGMP level were revealed in both cases. Changes in the content of cyclic nucleotides were followed by marked changes in their physiologically important ratios.     

Cyclic nucleotides in experimental glaucoma

cAMP and cGMP contents were studied in various eye tissues of rabbits with experimental glaucoma induced by chronic intravenous adrenaline administration. Cyclic nucleotide level was measured in the retina, choroid, iris and ciliary body. An increase in the tissue cAMP level was found especially in the iris and ciliary body. An increase in tissue cAMP content is explained by an enhanced beta-adrenergic regulation in the eyes of rabbits with experimental glaucoma. No consistent changes were found in cGMP content in eye tissues.     

On the role of serotonin and 5-methoxy-tryptamine in the regulation of cell division in sea urchin eggs

The presence of serotonin in sea urchin eggs has been ascertained by high-performance liquid chromatography and thin-layer chromatography analysis of tissue-free bioamines. The results show the presence of both serotonin and 5-methoxytryptamine. The role of these substances in the cell division process has been studied by using the serotonin antagonists, gramine and metergoline. Both antagonists cause a significant delay of the cell division which, however, can be prevented by the addition of either 5-hydroxytryptophane, serotonin, or 5-methoxytryptamine. The effect of gramine on the different stages of the cell division process has also been investigated. Neither S phase nor mitosis are affected by the serotonin antagonist, while cleavage is delayed. The effect of serotonin seems mediated by calcium ions and cAMP. Gramine causes a marked increase in radio-calcium efflux from the fertilized egg, and at the same time lowers the cAMP level.