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.     

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.     

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.     

The role of cyclic nucleotides and prostaglandins in heart function.

A short review of the role of cyclic nucleotides and prostaglandins (PGs) in normal and pathological functions of the heart is given. Possible interrelationships of these two regulatory systems have been studied by using spontaneously beating rat atria preparations. Addition of noradrenaline (NA) to the incubate (1 . 10(-6) M) caused an increase in amplitude and frequency which was preceded and parallelled by an elevation of the tissue cAMP level. A transient increase in cGMP and PGE values was also seen. Propranolol (5 . 10(-6) M) abolished the increase in amplitude and frequency as well as in cAMP and PGE concentrations. Indomethacin (1 . 10(-5) M) inhibited the formation of PGE. The increase in cGMP was blocked by phenoxybenzamine. Interchange between beta- and alpha-receptors according as the temperature is lowered has been described earlier. Hypothermia (20 degrees C) had a positive inotropic effect on the atria and increased the tissue cAMP concentration. Loading of the atria caused an increase in cAMP without any effects on cGMP or PGs. Slight hypoxia did not change the cAMP or PG levels, but elevated the cGMP values. Arrhythmias induced by hypo- or hyperpotassemia did not modify the biochemical parameters measured. PGF2alpha (1. 10(-5) M) normalized the atrial rhythm and increased the amplitude without changing cyclic nucleotide or PG levels. PGE1 (1 . 10(-4) M) increased the amplitude of normorhythmic atria and the tissue concentration of cAMP. PGE2 was the only PG tested which stimulated the heart adenylate cyclase in vitro. There seems to be close but complicated relationships between cyclic nucleotides and PGs in the heart.     

Substrate and effector specificity of a guanosine 3':5'-monophosphate phosphodiesterase from rat liver.

Guanosine 3':5'-monophosphate phosphodiesterases, which appear to be under allosteric control, have been partially purified from rat liver supernatant and particulate fractions. The preferred substrate for both phosphodiesterases was cGMP (Km values: cGMP less than cIMP less than cAMP). At subsaturating concentrations of substrate, the phosphodiesterases were stimulated by purine cyclic nucleotides. The order of effectiveness for activation of cyclic nucleotide hydrolysis was cGMP greater than cIMP greater than cAMP greater than cXMP. Using cAMP derivatives as activators of cIMP hydrolysis, modifications in the ribose, cyclic phosphate, and purine moieties were shown to alter the ability of the cyclic nucleotide to activate the supernatant enzyme. cGMP, at concentrations that stimulated cyclic nucleotide hydrolysis, enhanced chymotryptic inactivation of the supernatant phosphodiesterase. At similar concentrations, cAMP was not effective. It appears that on interaction with appropriate cyclic nucleotides, this phosphodiesterase undergoes conformational changes that are associated with increased catalytic activity and enhanced susceptibility to proteolytic attack. Divalent cation may not be required for the nucleotide-phosphodiesterase interaction and resultant change in conformation.     

Cyclic nucleotides in the rat brain in the dynamics of hypothalamic self-stimulation

The content of cyclic nucleotides was measured in the brain structures of rats performing hypothalamic self-stimulation. Changes of the cAMP content were shown to possess a specific pattern corresponding to the features of self-stimulation. An increasing self-stimulation frequency (SSF) was followed by the maximum increase in the above index in the sensorimotor cortex; a stable SSF was accompanied by activation of the cAMP-dependent mechanisms of the septum, hypothalamus, and posterior hypophysis, while a decreasing SSF correlated with suppression of these processes in the septum, hypothalamus, and anterior hypophysis. Changes in the cAMP phosphodiesterase activity were less intensive and did not depend on the cGMP level. Changes in the cGMP content were nonspecific and unidirectional: this index increased, with the maximum at the stable self-stimulation mode. The ratio of cyclic nucleotides shifted toward cAMP when the SSF increased, and towards cGMP when the SSF decreased, while at a stable frequency their content was equal to the control level. The changes in the cyclic nucleotide contents were non-reciprocal (except an inverse correlation between their contents in the hypothalamus observed when the SSF dropped).     

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.     

Immunocytochemical localization of cAMP and cGMP in cells of the rat carotid body following natural and pharmacological stimulation

Summary Although the chemoreceptive function of the carotid body has been known for many decades, the cellular mechanisms of sensory transduction in this organ remain obscure. Common elements in the transductive processes of many cells are the cyclic nucleotide second messengers, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Studies from our laboratory have revealed stimulus-induced changes in cyclic nucleotide levels in the carotid body as measured by RIA, but such changes in second messenger levels have not been localized to specific cellular elements in the organ. The present immunocytochemical study utilized the avidin-biotin-peroxidase method to investigate the distribution of cAMP and cGMP in the rat carotid body and to assess changes in the intensity of immunostaining following in vitro stimulation by hypoxia, forskolin, sodium nitroprusside, high potassium, and atrial natriuretic peptide. Both cAMP and cGMP immunoreactivity were localized to type I cells of organs maintained in vivo and fixed by perfusion. Organs exposed to 100% O₂-equilibrated media in vitro produced low but visible levels of cAMP immunoreactivity in a majority of type I cells; hypoxia (5% O₂-equilibrated media) for 10 min moderately increased the level of immunoreactivity; forskolin (10^–5 M), or forskolin combined with hypoxia, dramatically increased cAMP levels in virtually all cells. Moderate levels of cGMP immunoreactivity in control carotid bodies in vitro were strikingly reduced by hypoxia; a significant increase in cGMP levels occurred following incubation in high potassium (100 mM), and under these conditions, the decrease in cGMP immunoreactivity with hypoxia was much more pronounced. The synthetic analog of atrial natriuretic peptide, atriopeptin III (10^–7 M), greatly elevated cGMP immunoreactivity in the type I cells. On the other hand, sodium nitroprusside (1 mM) elevated cGMP staining mostly in vascular elements of the carotid body in vitro. The data implicate the involvement of cyclic nucleotides in transduction of natural chemosensory stimuli by the type I cells in rat carotid body.     

Cyclic nucleotides in ascites from ovarian carcinoma

Cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) were assayed in ascitic fluid from 27 patients with ovarian carcinoma and 23 patients with liver cirrhosis. The value of these cyclic nucleotides was correlated with standard methods for the clinical evaluation of tumors. No change in the cGMP levels was found in either of these groups. The cAMP content, however, was increased in 23 of the 27 cases of ovarian carcinoma. The high cAMP level was correlated with the cytological findings in only 13 (48.1%) of these cases.     

Immunocytochemical localization of cAMP and cGMP in cells of the rat carotid body following natural and pharmacological stimulation.

Although the chemoreceptive function of the carotid body has been known for many decades, the cellular mechanisms of sensory transduction in this organ remain obscure. Common elements in the transductive processes of many cells are the cyclic nucleotide second messengers, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Studies from our laboratory have revealed stimulus-induced changes in cyclic nucleotide levels in the carotid body as measured by RIA, but such changes in second messenger levels have not been localized to specific cellular elements in the organ. The present immunocytochemical study utilized the avidin-biotin-peroxidase method to investigate the distribution of cAMP and cGMP in the rat carotid body and to assess changes in the intensity of immunostaining following in vitro stimulation by hypoxia, forskolin, sodium nitroprusside, high potassium, and atrial natriuretic peptide. Both cAMP and cGMP immunoreactivity were localized to type I cells of organs maintained in vivo and fixed by perfusion. Organs exposed to 100% O2-equilibrated media in vitro produced low but visible levels of cAMP immunoreactivity in a majority of type I cells; hypoxia (5% O2-equilibrated media) for 10 min moderately increased the level of immunoreactivity; forskolin (10(-5) M), or forskolin combined with hypoxia, dramatically increased cAMP levels in virtually all cells. Moderate levels of cGMP immunoreactivity in control carotid bodies in vitro were strikingly reduced by hypoxia; a significant increase in cGMP levels occurred following incubation in high potassium (100 mM), and under these conditions, the decrease in cGMP immunoreactivity with hypoxia was much more pronounced.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hyperkalemia, hyperglycemia, and plasma levels of cyclic AMP and cyclic GMP induced by portal vein injection of cyclic nucleotides and their butyryl derivatives into dogs

The levels of serum potassium, blood glucose, and plasma adenosine cyclic 3':5'-monophosphate (cAMP) and guanosine cyclic 3':5'-monophosphate (cGMP) were studied after the portal vein injection of cyclic nucleotides and their derivatives, (cAMP, cGMP, N6, O2'-dibutyryl adenosine 3':5'-monophosphate (DBcAMP), N6-monobutyryl adenosine cyclic 3':5'-monophosphate (NMBcAMP), and O2'-monobutyryl adenosine cyclic 3':5'-monophosphate (OMBcAMP), into dogs. Dose-related hyperglycemic responses were observed after the injection of DBcAMP (1-8 mg/kg). Transient and prominent hyperkalemia and hyperglycemia were caused by the injection of DBcAMP, NMBcAMP, and OMBcAMP (4 mg/kg). The hyperkalemic response was highest with NMBcAMP (1.22 mequiv./L), followed by OMBcAMP (0.64), DBcAMP (0.54), cGMP (0.47), and cAMP (0.41), whereas the hyperglycemic response was highest with NMBcAMP (146 mg/100 mL), followed by DBcAMP (93.6), OMBcAMP (77.1), and cAMP (56.0), and there was only a slight change with cGMP (28.4) compared with the control. The plasma level of cAMP was maximal with DBcAMP (1.92 nmol/mL), followed by NMBcAMP (1.28) and OMBcAMP (0.76), whereas the plasma levels of cGMP showed no evident change, except that caused by DBcAMP (0.27). Of the cyclic nucleotides tested, NMBcAMP was found to be most potent in causing both hyperkalemia and hyperglycemia. Based on these results, possible correlations between hyperkalemia, hyperglycemia, and plasma levels of cAMP and cGMP are discussed.     

Role of cyclic nucleotides in modulating ovarian hCG action.

The role of cyclic nucleotides in mediating hormonally responsive adenylate cyclase and cAMP-dependent protein kinase was examined in vivo and in vitro when pseudopregnant rats were injected with hCG. Intracellular ovarian levels of cAMP increased, as expected, but no change in cGMP concentrations was observed. However, both cGMP and cAMP activated ovarian CDPK holoenzyme in vitro but cGMP had a lower affinity. The subunits of hCG were without effect. Even though cGMP and cAMP dissociate partially purified ovarian CDPK holoenzyme in vitro, the receptor sites of the regulatory subunit of CDPK would appear to be relatively specific for cAMP. Moreover, cGMP probably does not mediate hCG action in vivo.     

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.     

cGMP and cAMP cause pulmonary vasoconstriction in the presence of hemolysate.

We recently reported that addition of a small amount of hemolysate to the salt solution that perfused isolated rat lungs hypersensitized the vasculature to subsequent additions of ANG II or exposure to hypoxia, and addition of NO gas (. NO) to the perfusate that contained hemolysate caused a strong vasoconstrictor rather than a vasodilator response. In the present study, we demonstrate that CO and the secondary messengers cGMP and cAMP (usually associated with vasodilation) exert similar effects in hemolysate-perfused lungs. Analogs of the cyclic nucleotides cGMP or cAMP (8-bromo-cGMP and dibutyryl-cAMP, respectively) caused profound vasoconstriction in the isolated rat lung perfused with a salt solution that contained hemolysate. The cGMP- or cAMP-analog-induced vasoconstriction was inhibited by chemically dissimilar Ca2+ antagonists, by the protein phosphatase inhibitor okadaic acid, and, to a lesser degree, by protein kinase inhibitor H-7. Antiphosphothreonine immunoblotting demonstrated that lungs perfused with hemolysate exhibit increased phosphorylation of several proteins. These data indicate that, in the presence of hemolysate, pulmonary vasculature responds to nominally vasodilatory stimuli, including analogs of cGMP and cAMP, with vasoconstriction rather than vasodilation. The importance of our finding is the paradoxical nature of the response to (analogs of) cyclic nucleotides because, to our knowledge, cyclic nucleotide-induced vasoconstriction has not been previously reported.     

组胺H1和H2受体在兔灌流肺低氧性肺动脉加压反应中的作用

本实验采用兔自身血液灌流原位肺,用含氧6％的氮氧混合气进行人工通气,在此基础上,分别用扑尔敏和西咪替丁阻滞肺血管的H_1和H_2受体,观察组胺受体在低氧性肺动脉加压反应(HPPR)中的作用,并测定肺流出血液中血浆cAMP和cGMP含量。结果表明,扑尔敏可使兔灌流肺HPPR减弱,西咪替丁则使之增强。提示组胺参与兔灌流肺的HPPR;H_1和H_2受体在兔的HPPR中以H_2受体的作用占优势;血浆环核苷酸含量变化,可能是兔低氧时肺动脉收缩及组胺发生影响的生化基础之一。     

Changes in the system of cyclic nucleotides in irradiated body tissues

It has been shown that the content of cAMP in the rat thymus, spleen, and liver 1 and 3 days after gamma-radiation with 7.5 Gy decreases, and that of cGMP increases. Analogous dynamics has been revealed when studying adenylate cyclase and guanylate cyclase activities. The activity of cAMP and cGMP phosphodiesterases increased during the first period of study but subsequently it showed no distinction from the initial data level. The revealed postradiation changes in the content of cyclic nucleotides seem to be basically caused by the cyclases activity alterations.     

Dynamics of the cAMP and cGMP content of the heart in transient coronary insufficiency in rats

Experiments made on 127 white random-bred male rats weighing 200 +/- 10 g with transitory coronary insufficiency (TCI) with varying duration of myocardial ischemia (MI) have revealed consistent changes in the heart cAMP and cGMP. During MI, there was a biphasic variation in the concentration of cyclic nucleotides: an initial appreciable increase in the concentration was replaced by its lowering. At the same time the time course of cGMP content was more mobile in nature as compared to cAMP Reperfusion made at an early period (within the first 40 min) did not normally bring about the normalization of heart content of cyclic nucleotides whose concentration time course depended on the duration of the preceding MI. The pattern of changes in the concentration of cyclic nucleotides in the heart in TCI correlated to a significant degree with the previously described time course of the activity of the sympath- and cholinergic mechanisms by which heart work, contractile function and rhythm are controlled during TCI.     

Cyclic nucleotides during chondrogenesis: concentration and distribution in vivo and in vitro

This study correlates endogenous levels of cAMP and cGMP with their immunohistochemical localization during chondrogenic differentiation of C57B1/6J mouse limb mesenchyme in vivo and in vitro. A transient decrease in cGMP but not cAMP was found from days 12 to 13 in vivo correlating with early stages of chondrogenesis in the developing limb. Intracellular levels of both cAMP and cGMP in high density limb mesenchyme cultures increased 25% after 24 hr in culture when aggregate and nodule formation was detectable. When cells were seeded at different initial plating densities to delay the onset of aggregate and nodule formation, increased levels of intracellular cAMP correlated temporally with the appearance of nodules. Both cyclic AMP and cGMP were immunohistochemically localized in perichondrial cells and chondrocytes in vivo and in vitro. Therefore, (1) cAMP levels correlated temporally with the appearance of chondrogenic cells and (2) cAMP and cGMP were immunohistochemically localized to chondrogenic cells. These data indicate that fluctuations of both cAMP and cGMP levels may be involved in limb cartilage differentiation. Although increases in both nucleotides were found to correlate with the onset of chondrogenesis in vitro, in vivo data suggest that the amount of cAMP relative to cGMP rather than the absolute amount of an individual cyclic nucleotide may be more significant in modulating differentiation.     

Post-radiation changes in cyclic nucleotide levels and cAMP and cGMP in chromatin from rat blood lymphocytes

The change of contents of cyclic nucleotides (cAMP and cGMP) in lymphocyte chromatin of rats blood under norm and after X-ray irradiating in doses 0.5; 1 and 7.76 Gy was established. As well the change of correlation of the concentration of cAMP and cGMP in analysis in 1, 6, 12, 24 and 48 h after irradiating of animals by X-ray in different dose intensivity were shown.     

Characterization of the MRP4- and MRP5-mediated transport of cyclic nucleotides from intact cells

Cyclic nucleotides are known to be effluxed from cultured cells or isolated tissues. Two recently described members of the multidrug resistance protein family, MRP4 and MRP5, might be involved in this process, because they transport the 3',5'-cyclic nucleotides, cAMP and cGMP, into inside-out membrane vesicles. We have investigated cGMP and cAMP efflux from intact HEK293 cells overexpressing MRP4 or MRP5. The intracellular production of cGMP and cAMP was stimulated with the nitric oxide releasing compound sodium nitroprusside and the adenylate cyclase stimulator forskolin, respectively. MRP4- and MRP5-overexpressing cells effluxed more cGMP and cAMP than parental cells in an ATP-dependent manner. In contrast to a previous report we found no glutathione requirement for cyclic nucleotide transport. Transport increased proportionally with intracellular cyclic nucleotide concentrations over a calculated range of 20-600 microm, indicating low affinity transport. In addition to several classic inhibitors of organic anion transport, prostaglandins A(1) and E(1), the steroid progesterone and the anti-cancer drug estramustine all inhibited cyclic nucleotide efflux. The efflux mediated by MRP4 and MRP5 did not lead to a proportional decrease in the intracellular cGMP or cAMP levels but reduced cGMP by maximally 2-fold over the first hour. This was also the case when phosphodiesterase-mediated cyclic nucleotide hydrolysis was inhibited by 3-isobutyl-1-methylxanthine, conditions in which efflux was maximal. These data indicate that MRP4 and MRP5 are low affinity cyclic nucleotide transporters that may at best function as overflow pumps, decreasing steep increases in cGMP levels under conditions where cGMP synthesis is strongly induced and phosphodiesterase activity is limiting.