Pool Of Cyclic AMP Research Articles

Dopamine D4 receptor activation controls circadian timing of the adenylyl cyclase 1/cyclic AMP signaling system in mouse retina

In the mammalian retina, dopamine binding to the dopamine D₄ receptor (D₄R) affects a light-sensitive pool of cyclic AMP by negatively coupling to the type 1 adenylyl cyclase (AC1). AC1 is the primary enzyme controlling cyclic AMP production in dark-adapted photoreceptors. A previous study demonstrated that expression of the gene encoding AC1, Adcy1, is downregulated in mice lacking Drd4, the gene encoding the D₄R. The present investigation provides evidence that D₄R activation entrains the circadian rhythm of Adcy1 mRNA expression. Diurnal and circadian rhythms of Drd4 and Adcy1 mRNA levels were observed in wild-type mouse retina. Also, rhythms in the Ca²⁺-stimulated AC activity and cyclic AMP levels were observed. However, these rhythmic activities were damped or undetectable in mice lacking the D₄R. Pharmacologically activating the D₄R 4 h before its normal stimulation at light onset in the morning advances the phase of the Adcy1 mRNA expression pattern. These data demonstrate that stimulating the D₄R is essential in maintaining the normal rhythmic production of AC1 from transcript to enzyme activity. Thus, dopamine/D₄R signaling is a novel zeitgeber that entrains the rhythm of Adcy1 expression and, consequently, modulates the rhythmic synthesis of cyclic AMP in mouse retina.

Essential roles of dopamine D4 receptors and the type 1 adenylyl cyclase in photic control of cyclic AMP in photoreceptor cells

Light and dopamine regulate many physiological functions in the vertebrate retina. Light exposure decreases cyclic AMP formation in photoreceptor cells. Dopamine D(4) receptor (D(4)R) activation promotes light adaptation and suppresses the light-sensitive pool of cyclic AMP in photoreceptor cells. The key signaling pathways involved in regulating cyclic AMP in photoreceptor cells have not been identified. In the present study, we show that the light- and D(4)R-signaling pathways converge on the type 1 Ca(2+)/calmodulin-stimulated adenylyl cyclase (AC1) to regulate cyclic AMP synthesis in photoreceptor cells. In addition, we present evidence that D(4)R activation tonically regulates the expression of AC1 in photoreceptors. In retinas of mice with targeted deletion of the gene (Adcy1) encoding AC1, cyclic AMP levels and Ca(2+)/calmodulin-stimulated adenylyl cyclase activity are markedly reduced, and cyclic AMP accumulation is unaffected by either light or D(4)R activation. Similarly, in mice with disruption of the gene (Drd4) encoding D(4)R, cyclic AMP levels in the dark-adapted retina are significantly lower compared to wild-type retina and are unresponsive to light. These changes in Drd4-/- mice were accompanied by significantly lower Adcy1 mRNA levels in photoreceptor cells and lower Ca(2+)/calmodulin-stimulated adenylyl cyclase activity in retinal membranes compared with wild-type controls. Reduced levels of Adcy1 mRNA were also observed in retinas of wild-type mice treated chronically with a D(4)R antagonist, L-745870. Thus, activation of D(4)R is required for normal expression of AC1 and for the regulation of its catalytic activity by light. These observations illustrate a novel mechanism for cross-talk between dopamine and photic signaling pathways regulating cyclic AMP in photoreceptor cells.

Mannosyl-phospho-dolichol synthase from Trichoderma reesei is activated by protein kinase dependent phosphorylation in vitro

Preincubation of microsomes from Trichoderma reesei QM 9414 with the catalytic subunit of cyclic AMP dependent protein kinase from bovine heart resulted in increased activity of the mannosylphospho-dolichol synthase (EC 2.4.1.83, ‘Dol-P-Man synthase’) present in the membranes. This effect decreased upon subsequent treatment of the microsomes with alkaline phosphatase. SDS-PAGE of microsomal proteins indicated the presence of a strong band at 32 kDa, which corresponds to the size of yeast Dol-P-Man synthase. Fluorography of microsomal proteins, after incubation with [γ-32P]ATP and protein kinase, revealed the incorporation of 32P into this 32-kDa as well as several other microsomal proteins. Dol-P-Man synthase activity, assayed at various times of growth, correlated with the rise and fall of the cyclic AMP pool in T. reesei. Addition of extracellular dibutyryl cyclic AMP and theophyllin, however, did not elevate the internal cyclic AMP pool in the fungus, and did not cause an effect on Dol-P-Man synthase activity.

Mannosyl-phospho-dolichol synthase fromTrichoderma reeseiis activated by protein kinase dependent phosphorylation in vitro

Abstract Preincubation of microsomes from Trichoderma reesei QM 9414 with the catalytic subunit of cyclic AMP dependent protein kinase from bovine heart resulted in increased activity of the mannosylphospho-dolichol synthase (EC 2.4.1.83, ‘Dol-P-Man synthase’) present in the membranes. This effect decreased upon subsequent treatment of the microsomes with alkaline phosphatase. SDS-PAGE of microsomal proteins indicated the presence of a strong band at 32 kDa, which corresponds to the size of yeast Dol-P-Man synthase. Fluorography of microsomal proteins, after incubation with [γ- 32 P]ATP and protein kinase, revealed the incorporation of 32 P into this 32-kDa as well as several other microsomal proteins. Dol-P-Man synthase activity, assayed at various times of growth, correlated with the rise and fall of the cyclic AMP pool in T. reesei . Addition of extracellular dibutyryl cyclic AMP and theophyllin, however, did not elevate the internal cyclic AMP pool in the fungus, and did not cause an effect on Dol-P-Man synthase activity.

Dopamine and its agonists reduce a light-sensitive poor of cyclic AMP in mouse photoreceptors

The exposure to bright light of dark-adapted (DKA) mouse retinas incubated in the dark (DI) in IBMX-containing medium causes a marked loss of cyclic AMP. This light response also occurs if the medium contains 10 mM aspartate or cobaltous ion, agents believed to confine the effects of light to photoreceptors. Thus, the action of light in the presence of either of these agents defines a light-sensitive pool of cyclic AMP in photoreceptors. This pool could also be reduced or eliminated in DKA-DI retinas by nanomolar to micromolar levels of dopamine (if the medium contained SCH23390, a potent antagonist of D1 receptors), thus indicating an agonistic action of dopamine at D2 receptors. The D2 agonists LY171555 (EC50 10 nM) or (+)-3-PPP also reduced the cyclic AMP level in the dark. Of the D2 antagonists tested, the butyrophenone spiperone (in the presence of the 5HT-2 blocker ketanserin) countered the action of the D2 agonists but substituted benzamides were ineffective. Consistently, the D2 agonists had no effect on cyclic AMP levels of mutant retinas lacking photoreceptors (rd/rd), but reduced cyclic AMP in DKA-DI glutamate-modified retinas which exhibit a major loss of inner retinal neurons without apparent loss of photoreceptors. The D1 antagonist SCH23390 only reduced cyclic AMP levels of DKA-DI retinas when cyclic AMP levels had been elevated by adding dopamine to the incubation medium.

Tryptamine and Some Related Molecules Block the Accumulation of a Light‐Sensitive Pool of Cyclic AMP in the Dark‐Adapted, Dark‐Incubated Mouse Retina

Dark-adapted retinas of mice (C57BL/6J) incubated in the dark in media containing 1 mM 3-isobutylmethylxanthine (IBMX) or 5 mM Co2+ accumulate cyclic AMP (cAMP). A portion of this pool is light sensitive, as light can prevent or reverse its accumulation. Similarly, tryptamine, serotonin, 5-methoxytryptamine, bufotenine, and 5-methoxydimethyltryptamine can block the accumulation of the light-sensitive pool of cAMP, whereas tryptophan, melatonin, N-acetylserotonin, 5-methoxytryptophol, and tetrahydro-beta-carbolines are inactive. The phenomenon is not seen with mutant mouse retinas (rd/rd), which lack most photoreceptors, but persists in abnormal retinas containing photoreceptors but with extensive neuronal depletion in the inner retina. Tryptamine also inhibits cAMP accumulation in either dark or light-adapted retinas exposed to forskolin alone but not in media containing high levels of forskolin plus 1 mM IBMX. There is some suggestion that serotonin 5-HT-2 antagonists can partially reverse the action of the tryptamines, but hitherto undescribed receptors may be involved. Current data suggest that photoreceptors are the target for the action of the tryptamines.

Accumulation of adenosine 3',5'-monophosphate in slices of rat cerebral cortex induced by alpha-adrenergic agonists. II. Studies on mechanisms underlying the interaction with adenosine.

Incubation of slices of rat cerebral cortex with the calcium ionophore A23187 produced small increases in the accumulation of adenosine 3',5'-monophosphate (cyclic AMP). While low concentrations of Ca2+ ions (e.g., 200 microM) were sometimes necessary, the presence of adenosine (e.g., 50 microM) was essential; no effect of ionophore was observed when isoproterenol or isobutylmethylxanthine was substituted for adenosine. These results are consistent with the previously advanced hypothesis that stimulation of alpha-adrenergic receptors in this issue may cause calcium mobilization and thereby produce a calmodulin-mediated stimulation of adenylate cyclase. However, there is no apparent explanation for the requirement for adenosine. In addition, the possibility that additional mechanisms may be operating was suggested by experiments in which the incorporation of 3H-adenine into cyclic AMP was examined under steady-state conditions. While brief exposure to 3H-adenine after maximal adenosine- or isoproterenol-induced accumulations had been achieved led to small increases in the specific activity of cyclic AMP, the combination of norepinephrine and adenosine (plus propranolol) produced substantial decreases in the specific activity of cyclic AMP. Since the rate of incorporation of radioactivity did not keep pace with the expansion of the cyclic AMP pool, it is possible that norepinephrine also caused some reduction in the rate of cyclic AMP degradation under these conditions. Other interpretations of these results are discussed.

Uptake of Cyclic AMP by Natural Populations of Marine Bacteria

The major objective of this study was to describe the mechanism(s) of cyclic AMP uptake by natural populations of marine bacteria. A second objective was to determine whether this uptake could contribute to the intracellular regulatory pool of cyclic AMP. Using high-specific-activity P-labeled cyclic AMP, we found several high-affinity uptake systems. The highest-affinity system had a half-saturation constant of <10 pM. This system was extremely specific for cyclic nucleotides, particularly cyclic AMP. It appeared to meet the criteria for active transport. Uptake of cyclic AMP over a wide concentration range (up to 2 muM) showed multiphasic kinetics, with half-saturation constants of 1 nM and greater. These lower-affinity systems were much less specific for cyclic nucleotides. Although much of the labeled cyclic AMP taken up by the high-affinity systems was metabolized, some remained as intact cyclic AMP within the cells during 1 h of incubation. This suggests that at least some of the bacteria use cyclic AMP dissolved in seawater to augment their intracellular pools.

Theoretical analyses of the functioning of the high- and low- K m cyclic nucleotide phosphodiesterases in the regulation of the concentration of adenosine 3′,5′-cyclic monophosphate in animal cells

The concentration of cyclic AMP in a cell is determined by the activities of three enzymes: adenylate cyclase; low- K m phosphodiesterase, and high- K m phosphodiesterase. Analysis of the properties of this system has been undertaken. When the system is treated as three soluble enzymes, the steady state concentration of cyclic AMP in the cell is seen to be relatively insensitive to the activity of high- K m diesterase, which is present in larger amounts than can be readily explained. By numerical simulation of a more realistic model which treats adenylate cyclase and low- K m diesterase as enzymes bound to the plasma membrane, it is demonstrated that the system can exhibit greater flexibility. The content of high- K m diesterase is shown to be sufficient to generate a concentration gradient of cyclic AMP in a cell where adenylate cyclase is on the plasma membrane. This lowers the steady state concentration of cyclic AMP. Although it is normally ineffective at controlling the cyclic AMP concentration near the membrane, the high- K m enzyme can be as effective as the low- K m diesterase at regulating the level further inside the cell. The rate of approach to a new steady state after modulation of cyclase activity is also increased by high- K m diesterase. Although only a single pool of cyclic AMP is assumed, differences are predicted between cyclic-AMP dependent processes at the plasma membrane compared with the interior of the cell. On or near the membrane, modulation of only the cyclase or low- K m diesterase can exert control on the process, and the rate of response will be faster than the rate of change of the cellular content of cyclic AMP. Nervous tissue may be an exception, where the high- K m diesterase does control the concentration near the membrane effectively.

Steroidogenic action of calcium ions in isolated adrenocortical cells.

The corticotropin-induced increase of total intracellular and receptor-bound cyclic AMP in isolated rat adrenocortical cells was strictly dependent on extracellular Ca(2+). A rise in bound cyclic AMP with rising Ca(2+) concentrations was accompanied by a decrease in free cyclic AMP-receptor sites. A Ca(2+)-transport inhibitor abolished the rise in bound cyclic AMP induced by corticotropin. These data suggested that during stimulation by corticotropin some Ca(2+) has to be taken up in order to promote the rise of the relevant cyclic AMP pool. In agreement with this view, adenylate cyclase activity from isolated cells proved also to be dependent on a sub-millimolar Ca(2+) concentration in the presence of corticotropin and GTP. When cells were treated under specific conditions, corticosterone production could be activated by Ca(2+) in the absence of corticotropin (cells primed for Ca(2+)). Ca(2+)-induced steroidogenesis of these cells, in the absence of corticotropin, was also accompanied by an increase in total intracellular and receptor-bound cyclic AMP, as was found previously with corticotropin-induced steroidogenesis in non-primed cells. Calcium ionophores increasing the cell uptake of Ca(2+) were not able, however, to increase the cyclic AMP pools in non-primed cells, unlike corticotropin in nonprimed cells or Ca(2+) in cells primed for Ca(2+). It was concluded that during stimulation by either corticotropin or Ca(2+) a possible cellular uptake of Ca(2+) must be very limited and directed to a specific site which may affect the coupling of the hormone-receptor-adenylate cyclase complex.

Regulation of lipogenesis in adipocytes independent effects of thyroid hormones, cyclic AMP and insulin on the uptake of deoxy- d-glucose

Thyroidectomy is known to enhance fat cell phosphodiesterase activity; as a result, the response to lipolytic hormones is markedly reduced. Thyroidectomy also stimulates overall lipogenesis and the uptake of glucose: the present experiments investigated whether there was a correlation between cyclic AMP and glucose uptake. The parameter measured was the transport and phosphorylation (uptake) of deoxy- d-glucose in the presence of two modifiers of the cyclic AMP pool: phosphodiesterase inhibitors and the analogue, dibutyryl cyclic AMP. The inhibition by methylxanthines and dibutyryl cyclic AMP of deoxy- d-glucose uptake observed, was the same in fat cells from normal and thyroidectomized rats: the latter nonetheless still maintained their enhanced glucose uptake. It was therefore concluded that thyroid hormones and cyclic AMP control this step by different, separate pathways. Insulin, well known for its lipogenic effect, enhanced deoxy- d-glucose uptake in fat cells from both normal and thyroidectomized rats to the same extent (about 40%). An additive effect of thyroidectomy and insulin on glucose uptake was thus demonstrated. These results imply that glucose uptake in the adipocyte is controlled by at least three factors: thyroid hormones, cyclic AMP and insulin, each of which can act independently. Maximum glucose uptake is achieved in the presence of a combination of low concentrations of cyclic AMP, of insulin, and in the absence of thyroid hormones.

Chlorpromazine and Hormonal Elevation of Cyclic AMP Contents in Turkey Erythrocytes and in Perfused Rat Heart and Liver

Abstract An investigation was carried out to determine whether chlorpromazine (CPZ) affected the elevation of cyclic AMP by isoprenaline in turkey erythrocytes and perfused rat hearts, and by glucagon in perfused rat liver. In the perfused heart the incorporation of 14C‐adenine (in the perfusate) into the cyclic AMP pool, was also measured. In turkey erythrocytes exposed to isoprenaline the cyclic AMP accumulation was markedly inhibited by CPZ at concentrations above 5 × 10‐5 M, whereas lower concentrations caused slight reduction of cyclic AMP elevation. In the perfused rat heart and liver CPZ, at concentrations compatible with the integrity of the organ, did not reduce or change the cyclic AMP increase after isoprenaline and glucagon, respectively. The results indicate that inhibition of adenylate cyclase activation is not a general feature of the membrane stabilizing effects of CPZ. The loss of cyclic AMP response in turkey erythrocytes exposed to high concentrations of CPZ may be related to impairment of the cell membrane. CPZ could not be used as a tool in an attempt to discriminate between cyclic AMP‐mediated and cyclic AMP‐independent hormone effects. The present results compared to results obtained by other investigators indicate that caution is needed in extrapolating from experiments done on cell fractions to intact tissue.

Responsiveness to glucagon in fetal hearts. Species variability and apparent disparities between changes in beating, adenylate cyclase activation, and cyclic AMP concentration.

Previous studies of the ability of the immature heart to respond to glucagon have yielded conflicting results. To test the possibility that the apparent discrepancies might be explained in part by species variability, isolated hearts of fetal mice and rats (13-22 days' gestational age) were studied under identical conditions in vitro. Changes in atrial rate and ventricular contractility were measured in spontaneously beating hearts exposed to glucagon, and activation of adenylate cyclase was assayed in cardiac homogenates. In mice of 16 days' gestational age or less, there was no change in heart rate in response to glucagon; at 17-18 days, minimal responsiveness was present; and after 19 days, 10muM glucagon caused an increase in spontaneous atrial rate of 30 +/- 4% (SEM) (P less than 0.001). Measurement of the extent and speed of volume displacement of the isotonically contracting hearts with a specially constructed capacitance transducer revealed that ventricular inotropic responsiveness also appeared after 17-19 days. Cardiac stores of glycogen were reduced in older hearts exposed to glucagon, but not in those aged less than 16 days. In contrast, glucagon failed to activate adenylate cyclase in homogenates of hearts of fetal mice at any age. Furthermore, glucagon failed to elicit an increase in the concentration of cyclic AMP in spontaneously beating hearts that developed tachycardia. Responses in hearts of fetal rats were distinctly different from those in mouse hearts: at no age was there any change in heart rate, strength of contraction, glycogen content, or adenylate cyclase activation. Thus, there are major species differences in cardiac pharmacological maturation. Although the mouse heart develops the ability to increase its rate and strength of contraction and to undergo glycogenolysis in response to glucagon well before birth, the rat heart does not. In addition, there is an apparent disparity in late fetal mouse hearts between the ability of glucagon to induce functional responses and its ability to stimulate adenylate cyclase and increase cyclic AMP levels. It is impossible, of course, to rule out absolutely the possibility that localized increases in a critical cyclic AMP pool were present but too small to measure in the entire tissue. Nevertheless, the most obvious interpretation of our results is that they are compatible with the hypothesis that glucagon may exert some of its hemodynamic effects independently from the adenylate cyclase-cyclic AMP system in the late-fetal mouse heart.

Kinetics of Adenosine 3′:5′‐monophosphate Accumulation in Dog Thyroid Slices

Dog thyroid slices have been stimulated in vitro by thyrotropin. The kinetics of adenosine 3':5'-monophosphate (cyclic AMP) accumulation due to the activation of adenylate cyclase were measured. Experimental results have been interpreted by means of a numerical simulation based on a theoretical description of the overall process which consists of three steps: (a) the penetration of thyrotropin in the slices, (b) the binding of the hormone to the specific receptor and the consequent activation of adenylate cyclase, (c) the kinetics of cyclic AMP accumulation in each cell due to the interplay between synthesis and degradation. The numerical values of the parameters needed for the simulation were deduced from separate experiments. Diffusion of thyrotropin in the slices has been calculated from the kinetics of efflux of [3H]sucrose, and with or without albumin. Adenylate cyclase activity and activation by thyrotropin in homogenates and purified membranes were measured. Binding experiments of cyclic AMP on crude extract of dog thyroid lead to the conclusion that the maximal capacity of the specific binding site is close to the cyclic AMP content in resting thyroid cells. The purpose of this paper is to verify the validity of the current concepts about the mechanisms taking place in this process by comparing experimental results and theoretical simulation. It results from the study that for this system the time between the activation of adenylate cyclase and half maximal cyclic AMPaccumulation is equal to 3 min 45 s and the pool of cyclic AMP is renewed three times per minute. It has been also stressed that the thickness of the slices as well as inhibitors of phosphodiesterases change the apparent kinetics of cyclic AMP accumulation.

Inhibition of peptide chain initiation in lysates from ATP-depleted cells I. Stages in the evolution of the lesion and its reversal by thiol compounds, cyclic AMP or purine derivatives and phosphorylated sugars

Anaerobic incubation of rabbit reticulocytes at 37°C in Krebs-Ringer solution supplemented with hemin but devoid of glucose resulted at the end of 1–2 h in a drastic decline of their ATP content and an attendant arrest of protein synthesis. Subsequent provision of glucose and reoxygenation of the cells was followed by a rapid replenishment of the ATP pool, while resumption of protein synthesis was markedly delayed. This lag period could be considerably reduced by addition of 5–10 mM adenine or 2,6-diaminopurine to the incubation medium. Lysates prepared from ATP-depleted cells exhibited disaggregation of the polysomes and an inhibition of the endogenously coded protein synthesis, when tested in a cell-free system supplied with an adequate ATP generator. Both alterations increased in severity with the progressive decay of the intracellular ATP pool. The early phase of partial inhibition following a 40–70% decrease of the cellular ATP level was fully reversible by fortifying the cell-free preparation with dithiothreitol or a suitable NADPH-generating system. Alternatively, the inhibition could be also overcome by millimolar amounts of adenine, 2,6-diaminopurine and a variety of other purine derivatives or cyclic AMP. The effect of these compounds was unrelated to the endogenous cyclic AMP pool. Joint addition of both dithiothreitol and cyclic AMP or adenine was necessary for relieving the initiation block in lysates derived from cells depleted of 80–90% of their ATP content. On further aggravating the conditions of energy starvation, an additional requirement for phosphorylated sugars, e.g. glucose 6-phosphate or fructose 1,6-diphosphate, became apparent. ATP depletion brought about by exposing the cells to Antimycin A or 2,4-dinitrophenol resulted in a lesion which was indistinguishable from that induced by anaerobic incubation. On the other hand, energy deprivation in cell-free lysates from untreated reticulocytes, preincubated in the absence of an ATP-generating system failed to duplicate the deleterious effect of intracellular ATP depletion. Some aspects bearing on the biochemical mechanism of the lesion and its reversal are discussed in the light of the available data.

Cyclic nucleotide phosphodiesterases and thyroid hormones.

Evidence is presented that modulation of the maximum velocity of a particulate low K-m cyclic adenosine 3':5'-monophosphate (cyclic AMP) phosphodiesterase by thyroid hormones is one mechanism for the regulation of the responsiveness of rat epididymal adipocytes to lipolytic agents such as epinephrine and glucagon. Fat cells of propylthiouracil-induced hypothyroid rats are unresponsive to lipolytic agents and the V-max of particulate low K-m cyclic AMP phosphodiesterase of these cells is elevated above normal. In vivo treatment of hypothyroid rats with triiodothyronine restores to control values both the lipolytic response of the fat cells to epinephrine and the V-max of the particulate bound low K-m cyclic AMP phosphodiesterase. No similar correlation is found with the soluble high K-m cyclic AMP phosphodiesterase. The phosphodiesterases of fat cells from normal and hypothyroid rats respond identically in vitro to propylthiouracil, triiodothyronine, methylisobutylxanthine, or theophylline, although the particulate low K-m cyclic AMP phosphodiesterase is inhibited to a greater extent than soluble cyclic guanosine 3':5'-monophosphate phosphodiesterase activity. Protein kinase of fat cells from hypothyroid rats can be stimulated by cyclic AMP to the same total activity as observed in fat cells of normal rats. However, less of the protein kinase in fat cells from hypothyroid rats was in the cyclic AMP-independent form. This shift in the equilibrium of protein kinase forms is consistent with an increased activity of low K-m cyclic AMP phosphodiesterase and probably results from a lowering of the lipolytically significant pool of cyclic AMP.

Location of an isoproterenol-responsive cyclic AMP pool in adrenergic nerve cell bodies and its relationship to tyrosine 3-monooxygenase induction.

To decide whether adenosine 3':5'-cyclic monophosphate (cyclic AMP) plays a role as a second messenger in the trans-synaptic induction of tyrosine 3-monooxygenase (EC 1.14.16.2), it is desirable to discriminate between neuronal and extraneuronal changes in cyclic AMP concentration. Treatment of newborn rats with nerve growth factor antiserum or 6-hydroxydopamine, leading to destruction of 61-85% of the adrenergic nerve cell bodies in the superior cervical ganglion, led to a decrease in cyclic AMP of only 16-28%. This observation demonstrates that a relatively small portion of cyclic AMP is localized in the adrenergic neurons. However, administration of isoproterenol produced an increase (12-fold) in cyclic AMP only in this neuronal pool. Neither single nor repeated injections of isoproterenol led to induction of tyrosine monoxygenase. This, together with previous observations that experimental conditions leading to induction of the enzyme do not produce significant increases of cyclic AMP in the whole ganglion, is taken as an indication that cyclic AMP is not acting as a second messenger in the trans-synaptic induction of tyrosine monooxygenase in the rat superior cervical ganglion. In the rat adrenal medulla, treatment with reserpine led to both a shortlasting (60-90 min) increase in cyclic AMP and a subsequent induction of tyrosine monooxygenase. However, the increase in cyclic AMP was almost completely prevented (40 compared to 320%) by pretreatment of the rats with propranolol while the induction of tyrosine monooxygenase was not diminished. This observation also argues against an exclusive key-function of cyclic AMP in trans-synaptic induction of tyrosine monooxygenase in the adrenal medulla.

Effect of adrenocorticotropic hormone on extracellular adenosine 3',5'-monophosphate in the hypophysectomized rat.

The effects of ACTH on cyclic AMP levels in peripheral and adrenal vein plasma as well as in adrenal tissue were studied in the hypophysectomized rat and correlated with the steroidogenic response. High doses of ACTH increased adrenal tissue cyclic AMP levels and also markedly elevated cyclic AMP in adrenal vein and peripheral plasma. The gradient between adrenal vein and peripheral plasma cyclic AMP following ACTH was comparable to that observed for corticosterone. Small doses of ACTH produced a maximal rate of steroidogenesis and a several fold elevation in adrenal tissue cyclic AMP, but produced only a slight change in adrenal vein plasma cyclic AMP and no detectable change in peripheral plasma cyclic AMP. The present study indicates that although the adrenal cortex can extrude cyclic AMP in response to ACTH, the gland probably contributes minimally to the extracellular cyclic AMP pool in rats under physiological conditions. (Endocrinology 92: 1502, 1973)

Formation of cyclic adenosine 3′,5′-monophosphate from adenosine in brain slices

Adenosine (0.1–1 mM) can elicit the formation of cyclic [ 14C]AMP from a pool of radioactive adenosine nucleotides derived by prior incubation of slices with [ 14C]adenine. Slices, pulse-labeled with a combination of 3 μM [ 3H]adenosine and 3 μM [ 14C]adenine ( 3 H 14 C = 9.2 , contain pool(s) of radioactive adenine nucleotides with a 3 H 14 C ratio of 6.7 ± 0.15 . After stimulation of cyclic AMP formation with histamine, adenosine or depolarizing agents, the cyclic AMP fraction of the adenine nucleotides has a 3 H 14 C ratio of 3.64 ± 0.12 suggesting that an identical pool of adenine nucleotides, serving as a precursor for cyclic AMP, has been labeled by adenine and adenosine, but that adenosine to a great extent labels another adenine nucleotide pool which does not serve as a precursor of cyclic AMP. When slices pulse-labeled with [ 14C]adenine are incubated with 0.1 mM [ 3H]adenosine, cyclic AMP is formed both from the precursor pool of cyclic AMP and from the adenosine. Thus, the elevated levels of cyclic AMP seen in brain slices after incubation with adenosine are due to both stimulation of an adenyl cyclase and to conversion of adenosine to cyclic AMP.