Separation of cyclic AMP and cyclic GMP from thymidine,electrolytes and polyvalent nucleotides in tissue samples

Cyclic AMP and cyclic GMP can be separated from thymidine and its possible metabolites, electrolytes, and polyvalent nucleotides using columns of acidic alumina. Electrolytes and thymidine are not adsorbed on acidic alumina at pH 4.4 while cyclic nucleotides and polyvalent nucleotides are adsorbed at this pH. Cyclic AMP and cyclic GMP are eluted together from acidic alumina with 0.2 M ammonium formate (pH 6.0) and the polyvalent nucleotides remain adsorbed. The cyclic nucleotides are separated by chromatography on Dowex AG 1 X 8 resin. Recovery is 60–64% for cyclic AMP and cyclic GMP isolated from renal tissue samples. This methodology permits the separation of tritiated thymidine from cyclic nucleotides which are present in tissue preparations used in studies on the role of cyclic nucleotides in cellular growth.     

Separation of cyclic AMP and cyclic GMP from thymidine, electrolytes and polyvalent nucleotides in tissue samples.

Cyclic AMP and cyclic GMP can be separated from thymidine and its possible metabolites, electrolytes, and polyvalent nucleotides using columns of acidic alumina. Electrolytes and thymidine are not adsorbed on acidic alumina at pH 4.4 while cyclic nucleotides and polyvalent nucleotides are adsorbed at this pH. Cyclic AMP and cyclic GMP are eluted together from acidic alumina with 0.2 M ammonium formate (pH 6.0) and the polyvalent nucleotides remain adsorbed. The cyclic nucleotides are separated by chromatography on Dowex AG 1 X 8 resin. Recovery is 60--64% for cyclic AMP and cyclic GMP isolated from renal tissue samples. This methodology permits the separation of tritiated thymidine from cyclic nucleotides which are present in tissue preparations used in studies on the role of cyclic nucleotides in cellular growth.     

Effects of decapitation, ether and pentobarbital on guanosine 3′,5′-phosphate and adenosine 3′,5′-phosphate levels in rat tissues

Cyclic GMP and cyclic AMP levels in eight different rat tissues were examined after animlas were immersed in liquid nitrogen. In order of decreasing concentration, cerebellu, kidney, lung and cerebral cortex contained the greatest quantities fo cyclic GMP. These tissues also contained relatively high concentrations of cyclic AMP. Compared to values in animals which were sacrificed in liquid nitrogen, levels of both nucleotides in many of the tissues examined were altered by decapitation or anesthesia with ether and pentobarbital. Decapitation increased the levels of both cyclic GMP and cyclic AMP in cerebellum, lung, heart, liver and skeletabl muscle. However, decapitation increased only cyclic AMP in cerebral cortex and kidney. Our previously reported high level of cyclic GMP in lung was attributed to ether anesthesia and surgical removal which increased the cyclic GMP content in lung, heart, testis and skeletal muscle. The effect of ether on cyclic GMP levels in lung and heart was blocked by pretreatment of animals with atropine which indicated that cholinergic agents increase cyclic GMP content in these tissues. Acetylcholine and carbachol in the presence of theophylline increased the accumulation of cyclic GMP in incubations of rat lung minces. Increases in cyclic GMP and cyclic AMP levels in cerebellum with ether anesthesia were prevented if rats were immersed in liquid nitrogen after anesthesis with ether. Anesthesia with pentobarbital decreased the levels of cyclic GMP in cerebellum and kidney and increased the nucleotide in heart, liver, testis and skeletal muscle compared to levels in tissues from animals immersed in liquid nitrogen. However, pentobarbital increased cyclic AMP levels in cerebellum and cerebral cortex and decreased the nucleotide in liver, kidney, testis and skeletal muscle. These studies provide a possible explanation for the variability in in vivo levels of cyclic GMP and cyclic AMP which have been previously reported. In addition, these studies support the hypothesis that the synthesis and degradation of cyclic AMP and cyclic GMP are regulated independently and not necessarily in a parallel or reciprocal manner. These studies also suggest that the increase accumulation of one cyclic nucleotide has no major effect on the synthesis and/or metabolism of the other; however, such interactions cannot be entirely excluded from the results of this study.     

The effects of hormonal and circadian cycles, stress, and activity on levels of cyclic AMP and cyclic GMP in pituitary, hypothalamus, pineal and cerebellum of female rats

Cyclic AMP and cyclic GMP levels were measured in the anterior and posterior pituitary, hypothalamus, pineal and cerebellum of female rats sacrificed during proestrus, metestrus and diestrus. In the first experiment rats were sacrificed by microwave irradiation between 0900 and 1100, between 1600 and 1800 and between 2100 and 2300. Cyclic AMP and cyclic GMP levels did not vary in any region tested as a function of the estrous cycle except for slightly elevated cyclic GMP levels in the posterior pituitary during proestrus. However the time of day at which the animals were sacrificed affected levels of cyclic AMP in the hypothalamus and cerebellum and levels of cyclic GMP in the cerebellum. In a second experiment female rats were all sacrificed between 2130 and 2330 during proestrus and diestrus. In this experiment rats were sacrificed either immediately upon removal from the home cage or after 10 min of immobilization stress, or after 10 min of open field activity. No differences in pituitary cyclic nucleotides were seen between proestrous and diestrous animals. However, stressed animals showed large cyclic AMP increases in the pituitary, and activity increased cyclic GMP levels in the cerebellum and pineal.     

An examination of possible mechanisms of angiotensin II-stimulated steroidogenesis.

Dog and rat adrenal glomerulosa cells and subcellular fractions have been utilized to evaluate the mechanism of angiotensin II- and angiotensin III-induced aldosterone production. The effects of angiotensin, ACTH, and potassium have been compared on cyclic AMP and cyclic GMP in isolated glomerulosa cells and adenylate cyclase activity in subcellular fractions. The effect of angiotensin II has also been assessed on Na+-K+-activated ATPase of plasma membrane enriched fractions of dog and rat adrenals. We have demonstrated no effect of angiotensin II or angiotensin III on either adenylate cyclase, cyclic AMP, cyclic GMP, or Na+-K+-dependent ATPase activity over a wide range of concentrations. Potassium ion in concentrations that stimulate significant aldosterone production was also without effect. The negative effects of angiotensin and potassium were contrasted against a positive correlation between an ACTH-induced effect on aldosterone production, adenylate cyclase, and cyclic AMP accumulation. These studies have served to demonstrate that neither adenylate cyclase, cyclic AMP, cyclic GMP, or Na+-K+-activated ATPase seem to be directly involved in the mechanism of action of angiotensins on aldosterone production in the rat and dog adrenal glomerulosa.     

Binding of cyclic AMP and cyclic GMP to renal cortical homogenates. Relationship with phosphorylation

This study examined the binding of both cyclic AMP and cyclic GMP to receptor proteins in particulate and soluble subfractions of renal cortical homogenates from the golden hamster. The binding of both nucleotides was compared to subsequent effects of both nucleotides on the phosphorylation of histone from identical fractions. Cyclic AMP binding and cyclic AMP-dependent protein kinase activity predominated in the cytosol, with some binding and enzyme activity also detected in particulate fractions. Cyclic GMP and cyclic GMP-dependent protein kinase activity could only be demonstrated in cytosolic fractions and represented only 20-30% of cyclic AMP-dependent activity in this fraction. Binding of both nucleotides was highly specific, however, cyclic AMP showed some interaction with cyclic GMP binding. Evidence suggesting that each nucleotide interacts with a specific protein kinase was as follows: both the binding activity of the cyclic nucleotides and their combined protein kinase activity show additivity; cyclic AMP and cyclic GMP binding activity could be separated on sucrose gradients; cyclic AMP and cyclic GMP protein kinase activity could be separated with Sephadex G-100 chromatography, after preincubation of homogenate supernatants with either cyclic AMP or cyclic GMP. The results demonstrate the presence of both cyclic AMP- and cyclic GMP-dependent protein kinase in renal cortex.     

Development of Adrenocortical Cyclic Nucleotide (Cyclic AMP and Cyclic GMP) and Corticosterone Orcadian Rhythms in Male and Female Rats

The daily rhythm of the adrenocortical cyclic nucleotides (cyclic AMP and cyclic GIMP) was studied in infant male and female Wistar rats before and after the establishment of an adult-like daily rhythm of plasma corticosterone. As in this strain the rhythm of corticosterone is known to be present on postnatal day 18, pups of 2 and 3 weeks of age were studied. The dams and the pups as well as the young adult animals were kept on a controlled 12L-12D photoperiod. Groups of 8-10 pups were killed at 4-hr intervals throughout the day. Plasma corticosterone levels and adrenal cyclic AMP and cyclic GMP concentrations were simultaneously measured and the daily patterns established. Pups of 2 weeks of age showed neither plasma corticosterone nor adrenal cyclic AMP rhythms whereas pups of 3 weeks of age exhibited a typical adult-like circadian rhythm for both variables. The patterns for adrenal cyclic GMP differed according to sex: In female pups no cyclic GMP circadian rhythm could be detected at either 2 or 3 wk. In male pups of 3 wk a typical mature rhythm for adrenal cyclic GMP was evident whereas in younger male pups (2 wk) a circadian rhythm was detected. This circadian rhythm, however, differed from mature circadian rhythm in that its peak was located at 1300 hr instead of 0700 hr. These results demonstrate that, unlike that of cyclic AMP the adrenal cyclic GMP circadian rhythm does not appear at the same time as the plasma corticosterone circadian rhythm. Moreover, a circadian rhythmicity for adrenal cyclic GMP can be found in the absence of any corticosterone circadian rhythm. These facts argue against the view of cyclic GMP being a mediator of ACTH-stimulated steroidogenesis.     

Development of Adrenocortical Cyclic Nucleotide (Cyclic AMP and Cyclic GMP) and Corticosterone Orcadian Rhythms in Male and Female Rats

The daily rhythm of the adrenocortical cyclic nucleotides (cyclic AMP and cyclic GIMP) was studied in infant male and female Wistar rats before and after the establishment of an adult-like daily rhythm of plasma corticosterone. As in this strain the rhythm of corticosterone is known to be present on postnatal day 18, pups of 2 and 3 weeks of age were studied. The dams and the pups as well as the young adult animals were kept on a controlled 12L-12D photoperiod. Groups of 8–10 pups were killed at 4-hr intervals throughout the day. Plasma corticosterone levels and adrenal cyclic AMP and cyclic GMP concentrations were simultaneously measured and the daily patterns established. Pups of 2 weeks of age showed neither plasma corticosterone nor adrenal cyclic AMP rhythms whereas pups of 3 weeks of age exhibited a typical adult-like circadian rhythm for both variables. The patterns for adrenal cyclic GMP differed according to sex: In female pups no cyclic GMP circadian rhythm could be detected at either 2 or 3 wk. In male pups of 3 wk a typical mature rhythm for adrenal cyclic GMP was evident whereas in younger male pups (2 wk) a circadian rhythm was detected. This circadian rhythm, however, differed from mature circadian rhythm in that its peak was located at 1300 hr instead of 0700 hr. These results demonstrate that, unlike that of cyclic AMP the adrenal cyclic GMP circadian rhythm does not appear at the same time as the plasma corticosterone circadian rhythm. Moreover, a circadian rhythmicity for adrenal cyclic GMP can be found in the absence of any corticosterone circadian rhythm. These facts argue against the view of cyclic GMP being a mediator of ACTH-stimulated steroidogenesis.     

Effects of adenosine 3':5'-cyclic monophosphate and guanosine 3':5'-cyclic monophosphate on 3H-uridine incorporation into glomerulosal cells of hypophysectomized rat adrenal cortex.

The effects of cyclic AMP and cyclic GMP on the incorporation of 3H-uridine into glomerulosal cells of hypophysectomized rats were investigated by high resolution autoradiography. The quantitative analysis of autoradiographs shows that cyclic nucleotides, like ACTH, enhance the tracer incorporation into both nuclear and mitochondrial compartments. These findings are discussed in relation to previous results indicating that both cyclic nucleotides function as intracellular mediators of the trophic action of ACTH on the rat adrenal zona glomerulosa. The hypothesis that the mechanism of the glomerulotrophic action of ACTH and cyclic nucleotides involves the stimulation of nuclear and mitochondrial RNA synthesis is discussed.     

ADRENAL MEDULLARY CYCLIC NUCLEOTIDE PHOSPHODIESTERASE.—REGULATORY PROPERTIES OF THREE ENZYME ACTIVITIES

Abstract— Cyclic nucleotide phosphodiesterase from bovine adrenal medulla was fractionated into multiple activities by two different procedures, sucrose gradient centrifugation and gel filtration. Extracts of frozen and thawed adrenal medulla homogenates gave two phosphodiesterase activity peaks following density gradient centrifugation. The higher molecular weight activity hydrolyzed both cyclic AMP and cyclic GMP; ethylene glycol-bis(aminoethyl ether)- N,N' -tetraacetic acid (EGTA) inhibited only the hydrolysis of cyclic GMP. The lower molecular weight activity hydrolyzed only cyclic AMP and was not inhibited by EGTA. The two activities were not interconverted by recentrifugation.
Gel filtration of cyclic nucleotide phosphodiesterase activity extracted from frozen and thawed adrenal medulla on Ultrogel AcA 34 resolved the enzyme into three distinct peaks of enzyme activity with molecular weights of 350,000 (Peak I), 229,000 (Peak II) and 162,000 (Peak III). The enzyme from fresh tissue was resolved into peak I and II and only a small fraction of Peak III. Peak I hydrolyzed both cyclic nucleotides, while peak II was a cyclic GMP-specific enzyme and peak III was specific for cyclic AMP. The hydrolysis of cyclic AMP by the activity in Peak I was markedly stimulated by cyclic GMP; the hydrolysis of cyclic GMP by peak II was inhibited by EGTA and stimulated by calcium and CDR (calcium-dependent regulator protein). Peak III, which appears to be particulate, is not activated by either cyclic GMP or calcium and CDR.     

Regional sensitivity of cyclic AMP and cyclic GMP in rat brain to central cholinergic stimulation

We determined cyclic AMP and cyclic GMP levels in 18 regions of rat brain following administration of two different centrally active cholinergic agonists. Administration of oxotremorine (2 mg/Kg IP), a muscarinic agonist, 10 minutes prior to sacrifice by exposure to high power microwave irradiation resulted in significant increases in cyclic GMP in cerebellum, brainstem, hippocampus, midbrain, thalamus and septal region. Cyclic AMP levels were significantly elevated in substantia nigra, nucleus interpeduncularis, hypothalamus, brainstem, midbrain and in the pituitary where a greater than tenfold increase was observed. Levels of plasma prolactin and corticosterone did not differ in any of the groups examined, but growth hormone was significantly lower in animals exposed to oxotremorine. Physostigmine (0.5 mg/Kg) a cholinesterase inhibitor, administered IP also produced elevations in cyclic AMP and cyclic GMP in several of the brain regions examined. These results indicate that multiple regions of the brain are responsive to central cholinergic activation of not only cyclic GMP, but also cyclic AMP system.     

The Influence of Hypoxia on the Concentrations of Cyclic Nucleotides in the Rat Brain

Abstract: In order to study the influence of hypoxia on cyclic nucleotides in the brain, we reduced arterial Po, for 15–30 min in lightly anaesthetised and artificially ventilated rats to obtain values ranging from about 45 to about 10 mm Hg. In an additional group (arterial Po₂ 18–22 mm Hg), the tissue hypoxia was aggravated by moderate arterial hypotension (mean arterial blood pressure about 80 mm Hg). In all animals, electrocortical activity was recorded. Cyclic GMP concentrations in cerebral cortex were unchanged in all groups but one. In that group, in which tissue hypoxia was severe enough to induce a suppression-burst EEG pattern and a measurable reduction in the adenylate energy charge, cyclic GMP concentrations were slightly increased ( p < 0.05). Cyclic AMP concentrations remained unaltered at all degrees of hypoxia studied. It is concluded that changes in cyclic nucleotides in brain tissue occur first at such severe degrees of hypoxia of the duration studied that function and metabolism are profoundly altered.     

Metabolic and cyclic nucleotide enzyme activities in muscle and nonmuscle cells of rat heart during perinatal development

Enzyme activities related to aerobic metabolism and cyclic nucleotides were evaluated in muscle and nonmuscle cells of rat heart. The perinatal period from weaning to adult was studied. Malate dehydrogenase, citrate synthase, and 3-hydroxyacyl-CoA dehydrogenase activities of nonmuscle cells equal or exceed muscle cell activities in the weanling heart. Aerobic enzymes remain unchanged in nonmuscle cells during growth; however, muscle cell activities are enhanced. Adenylate cyclase and guanylate cyclase activities are higher in heart homogenates of weanling than adult rats. Despite elevated adenylate cyclase activity, cyclic AMP levels are identical in weanling and adult rats. Cyclic GMP levels are twofold higher in weanling than in adult rats. Muscle cell metabolism and cyclic nucleotide levels are associated with growth-related changes in heart function and cellularity, respectively.     

Changes in protein kinase activities during 2-acetylaminofluorene-induced hepatocarcinogenesis in the rat

Livers of rats fed the carcinogen 2-acetylaminofluorene (AAF) at a concentration of 0.025% were analyzed for protein kinase activities with [gamma 32P]ATP as substrate and either endogenous or exogenous (casein or histone) protein acceptors both in the presence or absence of cyclic nucleotides. Total protein kinase activity of the nuclear fraction, with exogenous histone or casein as substrate, was elevated during the first week of carcinogen administration. Total cytoplasmic kinase activities exhibited a pattern of activity change with maxima at about 25 and 42-49 days after the onset of carcinogen administration. Cyclic AMP levels rose steadily to approximately a 4-fold elevation by day 49 in livers of animals receiving carcinogen with the increase beginning prior to the development of externally visible nodular hyperplastic lesions. The findings demonstrate consistent and reproducible patterns of change in protein kinase activities that accompany AAF-induced hepatocarcinogenesis in the rat and provides the basis for a more detailed investigation of specific kinases.     

Cyclic nucleotide levels during carbachol-induced smooth muscle contractions.

Cyclic nucleotide levels and tension were measured at various times during carbachol-induced smooth muscle contractions. Cyclic GMP levels were markedly increased during contractions of rat vas deferens, guinea pig myometrium and guinea pig taenia coli, but were unchanged during contractions of rat uterus or guinea pig ileum. No significant changes in cyclic GMP levels could be detected in estrogen-primed rat uteri at any of the times or drug concentrations studied. Even in tissues in which large increases in cyclic GMP levels could be detected during carbachol-induced contractions (i.e. guinea pig myometrium and taenia coli) the contractions appeared to precede the cyclic GMP increases by several seconds. No significant changes in cyclic AMP levels were observed during carbachol-induced contractions in any of the smooth muscles studied. Thus, changes in tissue levels of the cyclic nucleotides do not appear to be responsible for the initiation of carbachol-induced smooth muscle contractions.     

Growth control in chick embryo fibroblasts; no evidence for a specific role for cyclic purine nucleotides

Cyclic AMP and cyclic GMP concentrations were measured in cultures of normal chick embryo fibroblasts and those transformed by Rous sarcoma virus under different growth conditions. No significant and reproducible correlation between the nucleotide levels and the rate of proliferation was observed. Neither release of normal cells from density dependent inhibition of growth nor transformation of the cultures by different strains of Rous sarcoma virus affected the concentrations of cyclic AMP or cyclic GMP. Activities of cellular cyclic nucleotide phosphodiesterases, enzymes involved in regulating the level of the nucleotides, were not directly affected by growth-stimulating concentrations of insulin or neuraminidase. Growth stimulation by insulin did not alter the activities of cellular cAMP-dependent protein kinase. These results do not support the hypothesis that cyclic AMP or cyclic GMP has a specific role in the growth control of chick embryo fibroblasts.     

Adenosine 3′,5′-monophosphate and guanosine 3′,5′-monophosphate: concentrations in Morris hepatomas of different growth rates

Cyclic AMP and cyclic GMP levels were examined in Morris hepatoma explants in vivo. All eight tumor lines examined had significantly elevated cyclic AMP and cyclic GMP levels when compared to normal liver from tumor-bearing rats. No apparent correlation was observed between the rates of tumor growth and cyclic nucleotide levels; however, two tumor lines (3924A and 7288ctc) had very high levels of cyclic GMP.     

CYCLIC NUCLEOTIDE PHOSPHODIESTERASE OF THE BOVINE RETINA: ACTIVITY, SUBCELLULAR DISTRIBUTION AND KINETIC PARAMETERS

Abstract— High phosphodiesterase activity for cyclic AMP and cyclic GMP was found in subcellular fractions of the bovine retina with more rapid hydrolysis of cyclic GMP than cyclic AMP in each fraction. Rod outer segments (ROS) and the supernatant fraction had highest activity. High enzyme activity remained associated with ROS membranes through several steps of purification by gradient centrifugation. A complex kinetic pattern was observed for cyclic AMP hydrolysis by the supernatant fraction yielding two values for K _m; a simple kinetic pattern was observed with cyclic GMP hydrolysis in supernatant and for both cyclic nucleotides in preparations of purified outer segments. Phosphodiesterase activity of outer segments was enhanced by Mg²⁺. Mn²⁺ and inhibited by EDTA. Cyclic AMP had relatively little effect on the hydrolysis of cyclic GMP in supernatant or ROS while cyclic GMP inhibited hydrolysis of cyclic AMP in both fractions.     

Effect of dexamethasone on steroidogenesis and on adrenocortical cyclic AMP and cyclic GMP responses to ACTH

The inhibitory action of dexamethasone on the adrenal steroidogenic response to ACTH was confirmed by im administration of graded doses (5, 10 and 30 ng) of synthetic beta 1-24 ACTH to young adult male rats which had received dexamethasone (0.1 mg/100 g bw) 4 hr prior to sacrifice. Following this, kinetic studies were performed by measuring plasma corticosterone, adrenocortical cyclic AMP and cyclic GMP before and 4, 12 and 30 min after administration of either 10 or 30 ng of ACTH. These doses were selected because their effects could be either completely or partially inhibited by dexamethasone. In rats without dexamethasone all the doses of ACTH which were checked induced an increase in both corticosterone and cyclic AMP and a decrease in cyclic GMP. With the smallest dose of ACTH the earlier administration of dexamethasone resulted in complete suppression of both the steroidogenic response and the cyclic AMP response. With the largest dose of ACTH both responses were diminished. In dexamethasone-treated rats the decrease in cyclic GMP was significantly less pronounced 4 min after ACTH than it was in non-treated rats. These results support the view that cyclic AMP and cyclic GMP might both be concerned with the mechanism of acute adrenal steroidogenesis.     

Ultrastructural localization of cyclic GMP and cyclic AMP in rat striatum

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