Adsorption chromatography of cyclic nucleotides on silica gel and alumina thin-layer sheets

Several solvent combinations of graded polarity have been developed, capable of separating cAMP or cGMP, not only from related nucleotides but from bases and nucleosides on alumina and silica gel thin-layer sheets (tls). The solvent system chloroform (C), methanol (M), water (W) (40:20:3) gave effective separations of cAMP on silica gel TLS. Identical results were obtained when this adsorption chromatography method was compared with the ion-exchange chromatography method involving PEI for its effectiveness in the separation of [α-³²P]cAMP formed from [α-³²P]ATP by a preparation of bovine sperm cells. In addition, this solvent system effectively separates cAMP from inosine, hypoxanthine, xanthine, and uric acid which may be useful in determinations of cAMP arising from ³H- or ¹⁴C-prelabeled ATP. Effective separation of cGMP on silica gel tls was accomplished with C:M:W (30:30:5). On alumina tls, cAMP and cGMP were separated from related compounds with M:W combinations; the solvent M:W (50:50) gave the lowest blanks (0.02%) for cGMP and it may prove useful in cGMP determinations.     

Gating of retinal rod cation channel by different nucleotides: Comparative study of unitary currents

Summary Single channels are observed after incorporation of native vesicles from bovine rod outer segment membranes into planar lipid bilayers. The activity of a single channel in the presence of cGMP is compared to that induced by the analog 8-bromo-cGMP and by cAMP. At +80 mV, K _0.5 is about 3 m for 8Br-cGMP, 18 m for cGMP and 740 m for cAMP. In cAMP, the amplitude of the current is smaller than in cGMP or 8Br-cGMP and depends on the filter cut-off frequency. The open/closed transition rates of the channel are slightly slower with 8Br-cGMP than with cGMP while they are 5 to 10 times faster with cAMP. Addition of Ni²⁺ ions to either cGMP or cAMP increases the open probability: the open/closed transition rates and amplitude of the current in cAMP are then comparable to those in cGMP. A dual effect of the addition of cAMP on the cGMPor 8Br-cGMP dependent activity previously reported (Furman, R.E., Tanaka, J.C. 1989. Biochemistry 28:2785–2788) is observed with a single channel: addition of subthreshold cAMP concentrations to cGMP (or to 8Br-cGMP) markedly increases P _o; addition of cAMP concentrations higher than about 70 m progressively accelerates the kinetics and reduces the amplitude to values observed in cAMP alone. The results are discussed in relation with the model previously proposed to account for the existence of four current levels (Ildefonse, M., Bennett, N. 1991. J. Membrane Biol. 123:133–147).     

Measurement of adenine nucleotides in plasma

The goal of this study was to identify the most important variables affecting bioluminescent ATP, ADP and AMP measurements in plasma and to develop an assay that takes these variables into account. Blood samples were drawn from conscious dogs. A ‘stop solution’ containing EDTA was prepared, which greatly retarded plasma ATP degradation by chelating Mg⁺² and Ca⁺² that are co‐factors for many ATPases. Stop solution and blood were mixed using a two‐syringe withdrawal system. Samples were centrifuged twice in order to remove red blood cells, and ATP was measured in the supernatant using the firefly luciferase assay. Sample pH was adjusted to the optimal range (7.75–7.95) and Mg²⁺ (necessary for the luciferase reaction) was added back to the sample within the luminometer 2 s prior to luciferase addition. Four assay tubes were prepared for each plasma sample, containing standard additions of 0–15 pmol added ATP, in order to quantify native plasma ATP content. In separate plasma/stop solution samples ADP + ATP was measured after converting ADP to ATP via the pyruvate kinase reaction, and AMP + ADP + ATP was measured after addition of both myokinase and pyruvate kinase. Addition of forskolin and isobutylmethylxanthine (IBMX) to the stop solution to inhibit platelets resulted in lower ATP concentrations. Measurement of ATP and haemoglobin from lysed erythrocytes revealed that haemolysis exerts a strong influence on plasma ATP concentration that must be taken into account. Copyright © 2003 John Wiley & Sons, Ltd.     

Absence of an effect of the lithium-induced increase in cyclic GMP on the cyclic GMP-stimulated phosphodiesterase (PDE II). Evidence for cyclic AMP-specific hydrolysis

Chronic treatment of rats with LiCl is known to induce a decrease in cAMP, while this decrease has also been found to occur together with both a simultaneous increase in total cortical phosphodiesterase (PDE; EC 3.1.4.17) activity and a concomitant increase in cGMP. These studies have implicated an involvement of PDE in lithium (Li⁺) action and it has been suggested that cGMP and the cGMP-stimulated PDE may be instrumental in the observed effects of Li⁺ on cAMP. In this study, three isozymes of PDE were isolated and identified from rat cortex and their activity determined, together with simultaneous measurement of cAMP and cGMP, after chronic treatment with oral LiCl (0.35% m/m). Li⁺ treatment exerted profound effects on cyclic nucleotides in the cortex, inducing significant suppression of cAMP while increasing cGMP levels. However, the ion only induced a slight but insignificant increase in the activities of the three PDE isozymes. To confirm these observations, methylparaben (MPB), a drug demonstrating both an ability to induce a selective stimulation of cAMP-specific PDE and also to lower intracellular levels of cGMP, was co-administration orally (0.4% m/m) with Li⁺ over the same period. This combination emphasized certain actions of Li⁺ not noted with Li⁺ alone. MPB inhibited the Li⁺-induced increase in cGMP, yet did not prevent the ion from decreasing cAMP. However, the combination of Li⁺ and MPB engendered a synergistic 100% increase in the activity of the membrane-bound, cAMP-specific PDE, PDE IV. This combination also produced a significant suppression of cAMP, while no reduction in cGMP was observed. The data is indicative that Li⁺-induced suppression of cAMP does not appear to be related to an effect on the cGMP-dependent PDE II, and that the increases in cGMP and PDE induced by Li⁺ observed previously and in the present study are two unrelated events. Instead, the synergistic response of Li⁺ plus MPB on PDE IV, and the associated reduction of cAMP, indicate that Li⁺ may promote selective cAMP hydrolysis via an effect on membrane-bound forms of PDE. This effect of Li⁺ on PDE IV, as well as the reciprocal effects on cyclic nucleotide balance, may have important implications in explaining the antipsychotic actions of the ion.     

Four cases of direct ion channel gating by cyclic nucleotides

Four different nucleotide-gated ion channels are discussed in terms of their biophysical properties and their importance in cell physiology. Channels activated directly by cGMP are present in vertebrate and invertebrate photoreceptors. In both cases cGMP increases the fraction of time the channel remains in the open state. At least three cGMP molecules are involved in channel opening in vertebrate photoreceptors and the concentration of the cyclic nucleotide to obtain the half maximal effect is about 15 µM. The light-dependent channel of both vertebrates and invertebrates is poorly cation selective. The vertebrate channel allows divalent cations to pass through 10–15-fold more easily than monovalent ions. In agreement with their preference for divalent cations, this channel is blocked byl-cis Dialtazem, a molecule that blocks certain types of calcium channels. In olfactory neurons a channel activated by both cAMP and cGMP is found and, as in the light-dependent channel, several molecules of the nucleotide are needed to open the channel with a half maximal effect obtained in the range of 1–40 µM. The channel is poorly cationic selective. A K⁺ channel directly and specifically activated by cAMP is found inDrosophila larval muscle. At least three cAMP molecules are involved in the opening reaction. Half-maximal effect is obtained at about 50 µM. This channel is blocked by micromolar amount of tetraethylammonium applied internally. Interestingly, this channel has a probability of opening 10–20-fold larger in the mutantdunce, a mutant that possesses abnormally elevated intracellular cAMP level, than in the wild type.     

An ATP-synthesizing system in seeds

Using onion seed powder, a semi-in vitro system for ATP synthesis in seeds has been developed. The system requires AMP, phosphoenolpyruvate (PEP) and orthophosphate with apparent Km values of 0.8, 1.5 and 3.0 mM, respectively. ATP synthesis is pH-dependent with a sharp optimum at pH 6.4, it exhibits linearity with time up to 40 min, and with a seed powder concentration between 25 and 150 mg ml^-1. The system is stimulated by low concentrations (<25 mM) of K⁺ and Mg²⁺ but is inhibited by higher concentrations of K⁺ and Mg²⁺ as well as by low concentrations of Li⁺, Na⁺ and especially Ca²⁺. The maximal rate is about 5 pmol min^-1 mg seed powder^-1 in dry onion seeds. During seed imbibition the rate of activity increases by about 120% after 3 h, reaching a plateau which is steady up to 18 h, when the radicle emerges. A comparison of the ATP content in seeds during the early period of imbibition with the capacity of ATP synthesis at this stage reveals that the described system could provide, during germination, 100 times more ATP than that found in imbibed seeds. The system is shown to be present in ten different types of seeds.     

Bidirectional effect of met-enkephalin on macrophage effector functions

Summary Met-enkephalin (ME) exerts a bimodal effect on functional activities of rat peritoneal macrophages (PM); in a range of low concentration (10^-9-10^-7 M) antibody dependent cellular cytotoxicity (ADCC)was markedly stimulated with a simultaneous decrease of Fc receptor (FcR) mediated phagocytosis while the opposite was observed at 10^-6-10^-5 M concentrations.Studying the possible underlying mechanism(s) the followings were recorded: (1) ME in all applied concentrations induced an early Na⁺ influx which was followed by a Ca²⁺ efflux in the range of low concentrations. In the range of high concentrations Na⁺ influx was accompanied by a Ca²⁺ influx. (2) ME at 10^-8 M concentration induced a rise in cGMP level with a plateau in the 60–120th min of incubation. This effect was prevented by 10^-5 M of naloxone. At 10^-6 M concentration a transient rise of cAMP level was recorded which was not affected by naloxone. (3) Verapamil in 10^-6 M abolished both the Ca²⁺ influx and the rise in cAMP level induced by 10^-6-10^-5 M ME but not the rise in cGMP level induced by lower ME concentrations. (4) cAMP elevation by high ME concentrations was abolished by enkephalinase inhibitory puromycin. (5) PM-enkephalinase as assessed by the cleavage of fluorogenic substrate L-alanine beta naphthylamide (ABNA), was inhibited by 10^-6-10^-5 M of ME. This inhibition was abolished by verapamil, but not affected by naloxone. In the range of low concentrations ME appears to act on specific delta opioid receptors and its action is positively coupled to guanylate cyclase. In relatively higher concentrations ME-action is not mediated by specific delta opioid receptors and it appears to involve Ca²⁺ influx, adenylate cyclase activation as well as the processing of hormone by PM-enkephalinase.     

Development of spatio-temporal pattern of Ca2+ on the chemotactic behaviour ofPhysarum plasmodium

Summary The development of a spatio-temporal pattern of Ca²⁺ concentration by a plasmodium ofPhysarum polycephalum during chemotaxis was studied using fura-2. Whenever the cell displayed coordinated migration in one direction as a whole body, a spatiotemporal pattern was established with a characteristic feature along the longitudinal axis. Calcium concentration oscillated with a period of a few minutes within the cell; the mean concentration at the front was higher than that at the rear. When the cell was given an attractant only at the rear end, the mean concentration rose at the site of application with an immediate increase in the frequency of oscillation. First, the change of the frequency is propagated toward the other end and then the mean level of the Ca²⁺ concentration at the non-stimulated site decreases. As a result, the Ca²⁺ gradient is reversed along the cell, which then begins to migrate in a coordinated manner in the reverse direction. This study showed that the spatiotemporal pattern of Ca²⁺ concentration is closely related to information processing for coordinated migration in chemotaxis. The role of the pattern in that process is discussed.     

Properties of calcium channels in cardiac muscle and vascular smooth muscle

The voltage-dependent slow channels in the myocardial cell membrane are the major pathway by which Ca²⁺ ions enter the cell during excitation for initiation and regulation of the force of contraction of cardiac muscle. The slow channels have some special properties, including functional dependence on metabolic energy, selective blockade by acidosis, and regulation by the intracellular cyclic nucleotide levels. Because of these special properties of the slow channels, Ca²⁺ influx into the myocardial cell can be controlled by extrinsic factors (such as autonomic nerve stimulation or circulating hormones) and by intrinsic factors (such as cellular pH or ATP level). The slow Ca²⁺ channels of the heart are regulated by cAMP in a stimulatory fashion. Elevation of cAMP produces a very rapid increase in number of slow channels available for voltage activation during excitation. The probability of a slow channel opening and the mean open time of the channel are increased. Therefore, any agent that increases the cAMP level of the myocardial cell will tend to potentiate I_si, Ca²⁺ influx, and contraction. The myocardial slow Ca²⁺ channels are also regulated by cGMP, in a manner that is opposite to that of CAMP. The effect of cGMP is presumably mediated by means of phosphorylation of a protein, as for example, a regulatory protein (inhibitory-type) associated with the slow channel. Preliminary data suggest that calmodulin also may play a role in regulation of the myocardial slow Ca²⁺ channels, possibly mediated by the Ca²⁺-calmodulin-protein kinase and phosphorylation of some regulatory-type of protein. Thus, it appears that the slow Ca²⁺ channel is a complex structure, including perhaps several associated regulatory proteins, which can be regulated by a number of extrinsic and intrinsic factors.VSM cells contain two types of Ca²⁺ channels: slow (L-type) Ca²⁺ channels and fast (T-type) Ca²⁺ channels. Although regulation of voltage-dependent Ca²⁺ slow channels of VSM cells have not been fully clarified yet, we have made some progress towards answering this question. Slow (L-type, high-threshold) Ca²⁺ channels may be modified by phosphorylation of the channel protein or an associated regulatory protein. In contrast to cardiac muscle where cAMP and cGMP have antagonistic effects on Ca²⁺ slow channel activity, in VSM, cAMP and cGMP have similar effects, namely inhibition of the Ca²⁺ slow channels. Thus, any agent that elevates cAMP or cGMP will inhibit Ca²⁺ influx, and thereby act to produce vasodilation. The Ca²⁺ slow channels require ATP for activity, with a K_0.5 of about 0.3 mM. C-kinase may stimulate the Ca²⁺ slow channels by phosphorylation. G-protein may have a direct action on the Ca²⁺ channels, and may mediate the effects of activation of some receptors. These mechanisms of Ca²⁺ channel regulation may be invoked during exposure to agonists or drugs, which change second messenger levels, thereby controlling vascular tone.     

Anoxia-induced changes in reactive oxygen species and cyclic nucleotides in the painted turtle

The Western painted turtle survives months without oxygen. A key adaptation is a coordinated reduction of cellular ATP production and utilization that may be signaled by changes in the concentrations of reactive oxygen species (ROS) and cyclic nucleotides (cAMP and cGMP). Little is known about the involvement of cyclic nucleotides in the turtle’s metabolic arrest and ROS have not been previously measured in any facultative anaerobes. The present study was designed to measure changes in these second messengers in the anoxic turtle. ROS were measured in isolated turtle brain sheets during a 40-min normoxic to anoxic transition. Changes in cAMP and cGMP were determined in turtle brain, pectoralis muscle, heart and liver throughout 4 h of forced submergence at 20–22°C. Turtle brain ROS production decreased 25% within 10 min of cyanide or N₂-induced anoxia and returned to control levels upon reoxygenation. Inhibition of electron transfer from ubiquinol to complex III caused a smaller decrease in [ROS]. Conversely, inhibition of complex I increased [ROS] 15% above controls. In brain [cAMP] decreased 63%. In liver [cAMP] doubled after 2 h of anoxia before returning to control levels with prolonged anoxia. Conversely, skeletal muscle and heart [cAMP] remained unchanged; however, skeletal muscle [cGMP] became elevated sixfold after 4 h of submergence. In liver and heart [cGMP] rose 41 and 127%, respectively, after 2 h of anoxia. Brain [cGMP] did not change significantly during 4 h of submergence. We conclude that turtle brain ROS production occurs primarily between mitochondrial complexes I and III and decreases during anoxia. Also, cyclic nucleotide concentrations change in a manner suggestive of a role in metabolic suppression in the brain and a role in increasing liver glycogenolysis.     

Soluble adenylyl cyclase regulates the cytosolic NADH/NAD+ redox state and the bioenergetic switch between glycolysis and oxidative phosphorylation

The evolutionarily conserved soluble adenylyl cyclase (sAC, ADCY10) mediates cAMP signaling exclusively in intracellular compartments. Because sAC activity is sensitive to local concentrations of ATP, bicarbonate, and free Ca²⁺, sAC is potentially an important metabolic sensor. Nonetheless, little is known about how sAC regulates energy metabolism in intact cells. In this study, we demonstrated that both pharmacological and genetic suppression of sAC resulted in increased lactate secretion and decreased pyruvate secretion in multiple cell lines and primary cultures of mouse hepatocytes and cholangiocytes. The increased extracellular lactate-to-pyruvate ratio upon sAC suppression reflected an increased cytosolic free [NADH]/[NAD⁺] ratio, which was corroborated by using the NADH/NAD⁺ redox biosensor Peredox-mCherry. Mechanistic studies in permeabilized HepG2 cells showed that sAC inhibition specifically suppressed complex I of the mitochondrial respiratory chain. A survey of cAMP effectors revealed that only selective inhibition of exchange protein activated by cAMP 1 (Epac1), but not protein kinase A (PKA) or Epac2, suppressed complex I-dependent respiration and significantly increased the cytosolic NADH/NAD⁺ redox state. Analysis of the ATP production rate and the adenylate energy charge showed that inhibiting sAC reciprocally affects ATP production by glycolysis and oxidative phosphorylation while maintaining cellular energy homeostasis. In conclusion, our study shows that, via the regulation of complex I-dependent mitochondrial respiration, sAC-Epac1 signaling regulates the cytosolic NADH/NAD⁺ redox state, and coordinates oxidative phosphorylation and glycolysis to maintain cellular energy homeostasis. As such, sAC is effectively a bioenergetic switch between aerobic glycolysis and oxidative phosphorylation at the post-translational level.     

The effects of cyclic nucleotides and some related agents on 32Pi labeling of renal polyphosphoinositides in vitro.

When rabbit kidney cortex slices were incubated in the presence of ³²P_i and dibutyrylcyclic AMP (dbcAMP)⁴ a significant decrease in the labeling of phosphatidyl inositol phosphate (DPI) but not phosphatidyl inositol bisphosphate (TPI) was observed. In the presence of 0.3 mm caffeine cyclic AMP (cAMP) produced a similar effect. Caffeine potentiated the inhibitory effect of dbcAMP. At high concentrations (3 mm) caffeine alone decreased the ³²P_i labeling of both DPI and TPI. These decreases in ³²P_i labeling were not mediated by decreases in the labeling of intracellular P_i or ATP as measured by 10-min acid-labile nucleotide phosphate (10′-ALNP). Addition of cyclic GMP (cGMP) to the incubation medium decreased the labeling of DPI and to a lesser extent that of TPI also. Addition of parathyroid hormone (PTH) to the incubation medium (in the absence of exogenous cyclic nucleotides) also decreased the ³²P_i labeling of DPI but not that of TPI. In contrast to the effects of cAMP, dbcAMP, cGMP, PTH, and caffeine, the addition of insulin to the incubation medium resulted in increased ³²P_i labeling of DPI with no effect on TPI labeling. DPI isolated from kidney cortex slices prelabeled with ³²P_i and subsequently incubated with cAMP or dbcAMP contained less label than DPI isolated from slices similarly prelabeled but subsequently incubated in the absence of either cAMP or dbcAMP. These data suggest an increased rate of DPI breakdown in the presence of elevated cAMP or dbcAMP concentrations. This hypothesis was supported by the fact that cAMP stimulated the hydrolysis of DPI but not of TPI by a polyphosphoinositide phosphodiesterase present in the supernatant fraction of rabbit kidney cortex.     

A sea urchin egg jelly peptide induces a cGMP-mediated decrease in sperm intracellular Ca(2+) before its increase

Speract, a sperm-activating peptide (SAP) from sea urchin eggs, increases the intracellular concentration of Ca²⁺ ([Ca²⁺]i) and modulates sperm motility. We measured the initial sperm response to speract using its caged analog and observed, for the first time, a small but significant decrease in sperm [Ca²⁺]i before the increase. Both directions of the [Ca²⁺]i change were completely blocked in high K⁺ seawater. Using membrane-permeant caged cyclic nucleotides (cNMP), only cGMP induced the decrease in [Ca²⁺]i although both cGMP and cAMP increased the [Ca²⁺]i. The decrease in the [Ca²⁺]i induced by cGMP was more notable following a second photolytic event, once [Ca²⁺]i had been elevated by an initial flash. This pattern of [Ca²⁺]i change was confirmed in individual sperm. These results together with pharmacological evidence suggest that the initial [Ca²⁺]i decrease is due to a Na⁺/Ca²⁺ exchanger activity, stimulated by hyperpolarization mediated by K⁺ efflux through cGMP-regulated K⁺ channels.     

Binding of [3H]phencyclidine to membranes from housefly thoracic muscles

The binding of [³H]phencyclidine ([³H]PCP) to a preparation of housefly thoracic muscle membranes was studied. Specific [³H]PCP binding saturated with both time and at concentrations of the radiolabeled ligand greater than 20 nM. One binding site with a K_D of 10.7 nM and a B_max of 3.9 pmol/mg protein was observed. Specific [³H]PCP binding was also readily reversible with a half-time of dissociation (t_1/2) of 13.8 min and varied proportionately with tissue concentration. Of the drugs tested, specific [³H]PCP binding was inhibited by PCP analogs, antipsychotics, antidepressants, Ca²⁺ channel antagonists, and K⁺ channel blockers. [³H]PCP binding was unaffected by addition of carbamylcholine or L-glutamate in absence or presence of ATP, GTP, cAMP, or cGMP. Though the identity of the [³H]PCP binding protein in housefly muscle membranes is still unclear, it is more likely to be an ionic channel such as K⁺ or Ca²⁺ channels than a neurotransmitter receptor.     

Cyclic nucleotides and haemoglobin concentration in rabbit bone marrow cell suspensions stimulated by purified erythropoietin

This study was conducted to determine the possible correlations between cyclic nucleotides cyclic adenosine monophosphate (cAMP) and cyclic guanine monophosphate (cGMP), and haemoglobin (Hb) concentration in nucleated cell suspensions of rabbit bone marrow incubated with erythropoietin (Ep). The levels of cAMP and cGMP were measured following the addition of different Ep concentrations to the suspensions. The Hb concentration was also measured in suspensions treated with Ep, dibutyryl cAMP (db-cAMP) or dibutyryl cGMP (db-cGMP), respectively. The following results were obtained: (1) upon the addition of 1 IU ml-1 Ep, an increase of cAMP levels was related to an increase in Hb concentration; while a decrease of Hb concentration was related to an increase of cGMP levels obtained when 0.1 IU ml-1 Ep was present in the incubation mixture. (2) A mimetic effect on Hb concentration was obtained upon the addition of db-cAMP or db-cGMP to the suspensions. (3) A quantitative correlation was found between the cAMP/cGMP ratio and Hb levels in cellular suspensions. This rapport was reviewed with respect to the controls as a decrease in Hb concentration when the ratio is less than one and an increase in Hb concentration when the ratio is greater than one.     

Second messengers and ion channels in acetylcholine-induced chloride secretion in hen trachea

1. Hen tracheal epithelium can be stimulated by serosal application of acetylcholine (ACh) to secrete Cl⁻ equal to ~ 60–90 μA/cm².2. Radio-ligand-displacement for IP₃, cAMP and cGMP and ion channel selective drugs in voltage clamp setups were employed to characterize second messengers and Cl⁻, K⁺ and Ca²⁺ channels involved in the ACh response.3. ACh induced a significant rise in IP, in isolated tracheocytes, while ACh did not influence the production of cAMP in whole tissue, isolated tracheocytes or basolateral cell membrane vesicles. Further ACh desensitization did not effect cAMP level in tracheocytes. In addition neither ACh stimulation nor desensitization interfered with cAMP production in presence of 4.5 μM forskolin in tracheocytes, a level of forskolin rising base level cAMP by around five fold.4. Around 35% of ACh Cl⁻ secretion depends on Ca²⁺ mobilization from internal stores and about 65% on Ca²⁺ influx over basolateral membrane. The activated Ca²⁺ channel is insensitive to class I, II, III and IV Ca²⁺ antagonists.5. A 23187 can mimic the ACh effect although 30% is indomethacin-sensitive demonstrating a prostaglandin activated adenylyl cyclase.6. Two K⁺ channels are involved in ACh secretion, one sensitive to Ba²⁺ and quinine and both insensitive to 4-aminopyridine, apamin, charybdotoxin and TEA.7. Flufenamate and triaminopyrimidine block a non-selective ion channel likely involved in the ACh response. An ACh activated apical Cl⁻ channel is NPPB-sensitive.     

Cyclic AMP,cyclic GMP,and bean rust uredospore germlings

Ten millimolar cyclic AMP (cAMP) or cyclic GMP (cGMP) induced bean rust uredospore germlings to undergo one round of mitosis and to form septa, processes normally associated with appressorium formation. To assess the possibility of cyclic nucleotide regulation of bean rust development, we used an 8-azido-[³²P]cAMP photoaffinity probe to identify three cyclic nucleotide binding peptides. The peptides bound either cAMP or cGMP. The phosphorylation of one peptide in uredospore germling extracts by [γ-³²P]ATP was stimulated by either 1 μM cAMP or cGMP, but only in the presence of 10 mM Na₂MoO₄, a phosphatase inhibitor. Uredospores contain about 1500 and 23 pmol cAMP and cGMP/g dry wt, respectively, as determined by radiobinding assays.     

Cyclic nucleotides and cell growth

Growth induction in resting fibroblast cultures by serum or growth factors induces a fast, transient cGMP peak which may constitute the intracellular signal for growth. A similar cGMP peak occurs when 3T3 cells arrested at the restriction point or in G₀ by starvation for certain amino acids are induced for growth by readdition of the lacking nutrients. Both 3T3 and SV3T3 cells which are arrested randomly all around the cell cycle do not exhibit major changes in cyclic nucleotides after growth induction. Determination of intracellular cAMP and cGMP levels in normal and transformed fibroblasts under different growth conditions shows that the transition between growing and resting state (G₀ arrest) is accompanied and probably induced by characteristic changes in cAMP to cGMP ratios. cGMP is decreased 2-5-fold in resting as compared to growing cultures, and increased 10-20-fold in activated cultures 20 min after serum induction. No major cGMP change was observed in growing, confluent, or serum-activated cultures of transformed cells. Measurement of guanylcyclase under unphysiological conditions (2 mM Mn⁺⁺) in crude and purified membranes from 3T3 and SV3T3 cultures did not show increased enzyme activity in the transformed cells. Significant differences may only show up when synchronized cells pass through the restriction point in G₁ phase. As a hypothesis it is proposed that transformed cells have an activated guanylcyclase system or a relaxed cGMP-pleiotypic response mechanism at the restriction point of their cell cycle.     

Mg2+ and ATP effects on K+ activation of the Ca2+-transport ATpase of cardiac sarcoplasmic reticulum

ATP and the divalent cations Mg²⁺ and Ca²⁺ regulated K⁺ stimulation of the Ca²⁺-transport ATPase of cardiac sarcoplasmic reticulum vesicles. Millimolar concentrations of total ATP increased the K⁺-stimulated ATPase activity of the Ca²⁺ pump by two mechanisms. First, ATP chelated free Mg²⁺ and, at low ionized Mg²⁺ concentrations, K⁺ was shown to be a potent activator of ATP hydrolysis. In the absence of K⁺ ionized Mg²⁺ activated the enzyme half-maximally at approximately 1 mM, whereas in the presence of K⁺ the concentration of ionized Mg²⁺ required for half-maximal activation was reduced at least 20-fold. Second MgATP apparently interacted directly with the enzyme at a low affinity nucleotide site to facilitate K⁺-stimulation. With a saturating concentration of ionized Mg²⁺, stimulation by K⁺ was 2-fold, but only when the MgATP concentration was greater than 2 mM. Hill plots showed that K⁺ increased the concentration of MgATP required for half-maximal enzymic activation approx. 3-fold.Activation of K⁺-stimulated ATPase activity by Ca²⁺ was maximal at anionized Ca²⁺ concentration of approx. 1 μM. At very high concentrations of either Ca²⁺ or Mg²⁺, basal Ca²⁺-dependent ATPase activity persisted, but the enzymic response to K⁺ was completely inhibited. The results provide further evidence that the Ca²⁺-transport ATPase of cardiac sarcoplasmic reticulum has distinct sites for monovalent cations, which in turn interact allosterically with other regulatory sites on the enzyme.