The influence of culture conditions on the induction of tyrosine aminotransferase by cyclic nucleotides in rat hepatoma cells.

The increase in tyrosine aminotransferase activity which occurs in rat hepatoma tissue culture (HTC) cells in response to cyclic AMP analogs has been shown to be an enzyme induction, similar to the larger response observed in certain other hepatoma cells and in liver. A specific antibody to tyrosine aminotransferase has been used to show that the number of enzyme molecules and the rate of enzyme synthesis are increased by N6,O2'-dibutyryl cyclic AMP in HTC cells. The effect on tyrosine aminotransferase is also produced by various 8-substituted derivatives of cyclic AMP and occurs whether or not the enzyme has been preinduced with a glucocorticoid. The response of the enzyme is greater when HTC cells are maintained in monolayer than in suspension cultures. Neither cell growth nor serum is required for the response.     

Isolation of multiple classes of mutants of CHO cells resistant to cyclic AMP

From mutagenized Chinese hamster ovary (CHO) cells we have isolated, in a single step, 11 independent mutants resistant to the growth-inhibitory effects of 8-Br-cyclic AMP, cholera toxin, and methylisobutylxanthine. Two major classes and several subclasses of mutants were obtained. Mutants from all classes have a normal doubling time. None of the mutants respond to cyclic AMP treatment with increased flattening and elongation as do the parental cells. Members of the first class have an altered protein kinase activity which has either an increased Ka for cyclic AMP or an absent response to cyclic AMP. Most of those mutations which result in a protein kinase with increased Ka for cyclic AMP (6/11) are dominant in somatic cell hybrids. Those mutations which result in a protein kinase with little or no response to cyclic AMP (3/11) are recessive. Members of the second major class (2/11) have normal levels of basal and cyclic AMP-dependent protein kinase activity. One is recessive and one is dominant by genetic tests. The basis for the defect in this second class of mutants has not been determined.     

Dibutyryl cyclic AMP resistant MDCK cells in serum free medium have reduced cyclic AMP dependent protein kinase activity and a diminished effect of PGE1 on differentiated function

Prostaglandin E1 (PGE1) has a stimulatory effect both on the growth and the expression of differentiated function of Madin Darby Canine Kidney (MDCK) cells in a hormonally defined medium (Medium K-1). While the stimulatory effect of PGE1 on MDCK cell growth is observed in subconfluent cultures, the effect of PGE1 on differentiated function (i.e., dome formation) is observed at confluency. PGE1 may possibly affect growth and such differentiated functions by separate mechanisms. In order to examine this possibility, dibutyryl cyclic AMP resistant variants of MDCK were selected. All of the variants were partially resistant to the growth inhibitory effects of dibutyryl cyclic AMP and theophylline. The cyclic AMP dependent protein kinase activity of four of the five variant clones studied was significantly reduced as compared with normal MDCK cells. The dependence of the kinase activity of several of the dibutyryl cyclic AMP resistant variants (DBr2 and DBr3) on the cyclic AMP concentration in the reaction mixture was compared with that of normal MDCK cells. At all of the cyclic AMP concentrations tested DBr2 and DBr3 cells had reduced protein kinase activity as compared with normal MDCK cells. This reduced activity could be attributed to a decrease in the Vmax for kinase in the two variants, rather than to a change in the Km of kinase for cyclic AMP. The cyclic AMP phosphodiesterase activity of dibutyryl cyclic AMP resistant variants was also studied. Unlike PGE1 independent clone 1, DBr2 and DBr3 cells did not differ significantly from normal MDCK cells with regard to their ability to degrade cyclic AMP. The growth and functional responsiveness of DBr2 and DBr3 cells to PGE1 was also examined. DBr2 and DBr3 cells were shown to retain a normal growth response to PGE1. However the capacity of DBr2 and DBr3 cells to form domes in response to PGE1 was dramatically reduced as compared with normal MDCK cells. Nevertheless DBr3 cells were shown to still retain the capacity to form domes in response to other inducers. The effect of PGE1 on one of the functional parameters involved in dome formation (the activity of the Na+/K+ATPase) was examined. The rate of ouabain-sensitive Rb+ uptake was observed to be elevated in confluent monolayers of normal MDCK cells maintained in Medium K-1, as compared with monolayers maintained in Medium K-1 minus PGE1.(ABSTRACT TRUNCATED AT 400 WORDS)     

Altered interaction between binding and catalytic subunits of a cyclic AMP-stimulated protein kinase from hepatoma cells.

Binding activity obtained from an established line of hepatoma tissue culture (HTC) cells has a lower apparent affinity for cyclic AMP at physiological pH than has the analogous binding activity from rat liver. However, the apparent binding affinity of HTC preparations can be reversibly increased by adding NaCl or guanidine · HCl. In the presence of such activating substances, a macromolecular inhibitory activity has been chromatographically separated from the cyclic AMP-binding activity. Removal of this inhibitory component causes the apparent affinity of the cyclic AMP-binding activity from HTC cells to increase and resemble that observed with liver preparations. Before treatment with salt, the inhibitory activity seems to be physically associated with the binding activity. Adding the isolated inhibitory component back to a suitably activated binding preparation from HTC cells results in a decrease in the apparent affinity for cyclic AMP. The isolated inhibitory component is devoid of cyclic AMP-binding and cyclic AMP phosphodiesterase activities and has an apparent minimal molecular weight of about 30,000 by gel filtration. It possesses protein kinase activity and seems to be identical to the catalytic subunit of a cyclic AMP-stimulated protein kinase on the basis of chromatographic properties and sensitivities to heat and low pH. This catalytic subunit represents only a minor portion of total cellular protein kinase activity and is also present in liver extracts. However, the binding activity from liver is not inhibited significantly under conditions where the binding from HTC cells is affected by the catalytic subunit. The difference in this inhibitory response between liver and HTC preparations appears to reflect differences in the cyclic AMP-binding proteins themselves.     

Influence of cyclic AMP on the growth response and anaerobic metabolism of carbon monoxide in Rhodocyclus gelatinosus

Rhodocyclus gelatinosus strain 1 (str. 1), a photoheterotrophic bacterium, used CO as an energy substrate under anaerobic CO/light conditions, and exhibited a diauxic growth response when CO was removed from the culture. Changes in the level of cyclic AMP which occurred in cells during diauxie suggested that the cyclic nucleotide operated as an intracellular control molecule. During CO/light-phase growth, intracellular cyclic AMP was 30 pmol/mg protein, and, as str. 1 adapted for photosynthetic growth after removal of CO, intracellular cyclic AMP levels decreased to 9 pmol/mg protein. Reexposure of a light culture to CO induced synthesis of CO oxidation activity (measured as CO:MV oxidoreductase). If 10 mM cyclic AMP was added with CO, the rate of synthesis of CO:MV oxidoreductase activity increased 25-fold, and str. 1 produced 1,230 units of activity (nmol CO oxidized min^-1 mg^-1 protein) after only 1 h. With cyclic AMP and no CO, no incerease in CO oxidation activity was seen. Appearance of CO oxidation activity in str. 1 represented de novo protein synthesis and was blocked with chloramphenicol. In addition to stimulating formation of CO oxidative activity, a high level of cyclic AMP in str. 1 during growth with CO appeared to influence photometabolism negatively by repressing bacteriochlorophyll formation.Abbreviations Bchl a bacteriochlorophyll a - MV methyl viologen - CO MV oxidoreductase, carbon monoxide: methyl viologen oxidoreductase     

Regulation of two phosphatases and a cyclic phosphodiesterase of Salmonella typhimurium.

The regulation of three Salmonella typhimurium phosphatases in reponse to different nutritional limitations has been studied. Two enzymes, an acid hexose phosphatase (EC 3.1.3.2) and a cyclic phosphodiesterase (EC 3.1.4.d), appear to be regulated by the cyclic adenosine 3' ,5'-monophosphate (AMP) catabolite repression system. Levels of these enzymes increased in cells grown on poor carbon sources but not in cells grown on poor nitrogen or phosphorus sources. Mutants lacking adenyl cyclase did not produce elevated levels of these enzymes in response to carbon limitation unless cyclic AMP was supplied. Mutants lacking the cyclic AMP receptor protein did not produce elevated levels of these enzymes in response to carbon limitation regardless of the presence of cyclic AMP. Since no specific induction of either enzyme could be demonstrated, these enzymes appear to be controlled solely by the cyclic AMP system. Nonspecific acid phsphatase activity (EC 3.1.3.2) increased in response to carbon, nitrogen, phosphorus, or sulfur limitation. The extent of the increase depended on growth rate, with slower growth rates favoring greater increases, and on the type of limitation. Limitation for either carbon or phosphorus resulted in maximum increases, whereas severe limitation of Mg2+ caused only a slight increase. The increase in nonspecific acid phosphatase during carbon limitation was apparently not mediated by the catabolite repression system since mutants lacking adenyl cyclase or the cyclic AMP receptor protein still produced elevated levels of this enzyme during carbon starvation. Nor did the increase during phosphorus limitation appear to be mediated by the alkaline phosphatase regulatory system. A strain of Salmonella bearing a chromosomal mutation, which caused constitutive production of alkaline phosphatase (introduced by an episome from Escherichia coli), did not have constitutive levels of nonspecific acid phosphatase.     

Glycosyl phosphatidylinositol (GPI)/inositolphosphate glycan (GPI): An intracellular signalling system involved in the control of thyroid cell proliferation

In porcine thyrocytes, TSH alone does not induce cell growth. Recently, it has been demonstrated that acute stimulation by TSH of porcine thyrocytes leads to release an inositolphosphate glycan (IPG) described as a putative second messenger for various growth factors in different cell types. IPG isolated from porcine thyrocytes induces proliferation of fibroblasts EGFR T17 and porcine thyrocytes. In porcine thyrocytes we have confirmed that cell growth requires the presence of both TSH and insulin. This effect is reproduced by 8-bromo cyclic AMP suggesting a mediation by intracellular cyclic AMP. Cooperative effects between 8-bromo cyclic AMP and IPG have also been evidenced and are in favour of a crosstalk between distinct signalling pathways.     

PGE1-independent MDCK cells have elevated intracellular cyclic AMP but retain the growth stimulatory effects of glucagon and epidermal growth factor in serum-free medium

Prostaglandin E1 (PGE1), a component in the hormone-supplemented, serum-free medium for the Madin Darby canine kidney (MDCK) cell line, has been proposed to increase MDCK cell growth by increasing intracellular cyclic AMP levels. The association between increased intracellular cyclic AMP and the growth stimulatory effect of PGE1 has been examined in normal MDCK cells and in PGE1-independent variants of MDCK. These variant cells have lost the PGE1 requirement for long term growth in defined medium. Normal MDCK cells had almost twofold higher intracellular cyclic AMP levels during growth in Medium K-1 (9.0 pmol/mg protein) than in Medium K-1 minus PGE1. Furthermore, PGE1-independent clone 1 had higher intracellular cyclic AMP levels in Medium K-1 minus PGE1 than normal MDCK cells in Medium K-1. This latter observation suggests that the PGE1 requirement for MDCK cell growth is associated with the low intracellular cyclic AMP levels of this cell line. An involvement of cyclic AMP in the growth response to PGE1 is supported by these observations, as well as by the growth stimulatory effects of other agents that affect cyclic AMP metabolism in MDCK cells. These agents include glucagon, isobutyl methylxanthine (IBMX), and dibutyryl cyclic AMP. The growth of PGE1-independent clone 1 was inhibited rather than stimulated by PGE1. Similarly, PGE1-independent cell growth was inhibited by IBMX and dibutyryl cyclic AMP. However, the growth response to one agent which increases cyclic AMP (glucagon) was retained in PGE1-independent clone 1. This result suggests that the effect of glucagon is not associated with increases in intracellular cyclic AMP. The growth stimulatory effect of epidermal growth factor (EGF) on normal MDCK cells was also studied. Although EGF does not act via a cyclic AMP-mediated mechanism, EGF increased normal MDCK cell growth and substituted for PGE1 in Medium K-1. Thus, EGF and PGE1 could possibly affect similar growth-related functions in MDCK cells, although by different pathways. This possibility was examined further, using PGE1-independent clone 1. EGF, like glucagon, was still growth stimulatory to the PGE1-independent cells. Consequently, the biochemical pathways by which EGF and PGE1 increase MDCK cell growth probably do not converge.     

Control of Photosynthetic Sucrose Synthesis by Fructose 2,6-Bisphosphate : III. Properties of the Cytosolic Fructose 1,6-Bisphosphatase

The cytosolic fructose 1,6-bisphosphatase from spinach (Spinacia oleracea U.S. hybrid 424) leaves has been partially purified and its response to fructose 2,6-bisphosphate, AMP, and fructose 1,6-bisphosphate studied, using concentrations present in the cytosol during photosynthesis. In the presence of fructose 2,6-bisphosphate, the substrate saturation kinetics for fructose 1,6-bisphosphate are sigmoidal, with half-maximal activity being attained in 0.1 to 1 millimolar concentration range. The inhibition is enhanced by AMP. Using these results, and information published elsewhere on metabolite concentrations, it is discussed how fructose 1,6-bisphosphatase activity will vary in vivo in response to alterations in the availability of triose phosphate and AMP, and the accumulation of the product, fructose 6-phosphate.     

Cyclic AMP-dependent and -independent effects on tissue-type plasminogen activator activity in osteogenic sarcoma cells; evidence from phosphodiesterase inhibition and parathyroid hormone antagonists

The plasminogen activator (PA) in clonal osteogenic sarcoma cells of rat origin (UMR 106-01 and UMR 106-06) and in osteoblast-rich rat calvarial cells has been characterized using specific antibodies to be tissue-type PA (tPA). An Mr value of 75,000 by SDS-polyacrylamide gel electrophoresis and fibrin autoradiography supports this characterization. There was also evidence for an Mr 105,000 component, which could be due to a proteinase-inhibitor complex. The mechanism of regulation of this tPA activity has been studied in the clonal osteogenic sarcoma cells. Parathyroid hormone (PTH) and prostaglandin E2, which increase cyclic AMP production in the sarcoma cells, also increased tPA activity. The sensitivity and magnitude of the tPA response to PTH and prostaglandin E2 were increased by simultaneous treatment with isobutylmethylxanthine (IBMX) at drug concentrations which had little effect themselves on tPA activity. In UMR 106-06 cells, which unlike UMR 106-01 cells show a cyclic AMP response to calcitonin, tPA activity was also increased in response to calcitonin, and the effect was enhanced by IBMX. 1,25-Dihydroxyvitamin D-3 also increased tPA activity in the cells, but this response was not modified by IBMX. Synthetic peptide antagonists of PTH-responsive adenylate cyclase, [34Tyr]-hPTH (3-34) amide and [34Tyr]-hPTH (5-34) amide, inhibited the PTH-induced increase in tPA activity over the same concentration range at which they inhibited cyclic AMP production, but the antagonist peptides had no effect on the tPA responses to prostaglandin E2, calcitonin or 1,25-dihydroxyvitamin D-3. These data indicate that cyclic AMP mediates the actions of PTH, prostaglandin E2 and calcitonin in increasing tPA activity in the clonal osteogenic sarcoma cells. 1,25-Dihydroxyvitamin D-3, on the other hand, increases tPA activity through a mechanism independent of cyclic AMP.     

Enhanced GTP-dependent activities of the adenylate cyclase system: basis for increased hormonal responsiveness.

Normal rat kidney (NRK) cells growth arrested by picolinic acid and isoleucine deprivation exhibit an increased response to certain agents (i.e., prostaglandin E₁, (?)-isoproterenol, and cholera toxin) which elevate intracellular cyclic AMP levels. The enhanced hormonal response is apparently due, at least in part, to increased adenylate cyclase activity. Adenylate cyclase activities measured in the presence of GTP, GTP plus prostaglandin E₁, and GTP plus (?)-isoproterenol are increased two- to threefold in membranes prepared from treated cells. In contrast, basal activity is potentiated only 20 to 50% and activity determined in the presence of fluoride is only marginally altered. Also of interest is the increase in cholera toxin activation of cyclase activity in the treated cells. Lower concentrations of cholera toxin (5 ng/ml) are required to achieve maximal stimulation of cyclase activity from picolinic acid-treated and isoleucine-deprived cells; maximal stimulation of control cell adenylate cyclase is attained with 25 to 50 ng/ml cholera toxin. Picolinic acid treatment and isoleucine deficiency both have been shown to arrest NRK cell growth in the G₁ phase of the cell cycle. However, results with cells arrested in G₁ by serum starvation and by growth to high cell population density indicate that G₁ specific growth arrest does not appear to account for the increase in hormonal responsiveness. Chelation of inhibitory metals and proteolytic activation also do not appear to be involved in the mechanism by which picolinic acid enhances cyclic AMP formation. Rather, the results suggest that the treated cells have an increased amount of an active GTP-dependent function required for hormone and cholera toxin stimulation of adenylate cyclase. Thus, picolinic acid treatment and isoleucine deprivation may provide a useful means of modulating the GTP-dependent step required to potentiate hormonal responsiveness.     

Mitotic and pigment-translocating activities of cultured chromatophores of the guppy, Lebistes reticulatus

Using Ham's F-12 medium, an in vitro culture system permitting cellular survival for over 6 months has been developed for the chromatophores of the guppy. In this culture system, the various types of chromatophores (melanophores, erythrophores and xanthophores) migrated out of the explanted tail fin tissue, retained their pigmentation, and displayed both mitotic and pigment-translocating activities. The mitotic activity was evident during the first 3 or 4 weeks in culture, whereas the pigment-translocating ability persisted for 16 weeks. The cultured chromatophores of male fish displayed pigment aggregation in response to adrenergic agents (epinephrine and norepinephrine) and pigment dispersion in response to alpha-melanocyte stimulating hormone (alpha-MSH), cyclic AMP and dibutyryl cyclic AMP. Cyclic GMP did not elicit pigment-translocating responses in any of the chromatophores.     

Action of the cardiac alpha 1-adrenergic receptor. Activation of cyclic AMP degradation

Using purified rat ventricular myocytes and membranes prepared from them, we have previously found that alpha 1-adrenergic stimulation causes decreased cyclic AMP accumulation and decreased activation of cyclic AMP-dependent protein kinase. We have now analyzed the mechanism by which alpha 1 stimulation is linked to cyclic AMP metabolism. In an adenylate cyclase assay in which carbachol inhibits the stimulatory effect of norepinephrine, the addition of prazosin (alpha 1-antagonist) has no effect on the response to norepinephrine. In membranes prepared from myocytes treated with pertussis toxin, norepinephrine competes for alpha 1-receptors (assessed by [3H]prazosin binding) with two components, binding to the high affinity component being sensitive to exogenous GTP, exactly as in membranes prepared from control myocytes. In intact cells labeled with [3H]adenine in which carbachol antagonizes the norepinephrine response, prazosin enhances accumulation of [3H]cyclic AMP due to norepinephrine. Treatment of cells with pertussis toxin eliminates inhibition by carbachol but does not alter prazosin's capacity to enhance the norepinephrine response. Addition of phosphodiesterase inhibitors eliminates this effect of alpha 1 blockade. In [3H]adenine-labeled cells loaded with [3H]cyclic AMP by prior treatment with isoproterenol, alpha 1-adrenergic stimulation enhances disappearance of [3H]cyclic AMP. Measurements of cellular cyclic AMP give results similar to those obtained with the adenine labeling technic. We conclude that occupation of the myocyte alpha 1-receptor results in stimulation of cyclic AMP phosphodiesterase activity.     

AMP deaminase in Dictycostelium discoideum: Increase in activity following nutrient deprivation induced by starvation or hadacidin

Summary AMP deaminase, the activity that catalyzes the deamination of AMP to form IMP and NH₃ has been measured in Dictyostelium discoideum. A new procedure to assay the activity of this enzyme was developed using formycin 5-monophosphate, a fluorescent analog of AMP as the substrate, and ionpaired reverse phase HPLC to separate the reactants and products. Quantitation of the formycin containing compounds was accomplished at 290 nm. At this wavelength adenosine containing compounds were not detected and activity could be monitored in the presence of its activator ATP. The AMP deaminase activity in vegetative cells was 7.4 nmols/min/mg proteins while the activity in cells measured at 2 and 6 hrs after starvation-induced growth-arrest was 376 nmols/min/mg protein... a 51-fold increase. When vegetative cells were treated with hadacidin, a drug that restricts de novo AMP synthesis and pinocytosis, the activity of the AMP deaminase was 511 nmols/min/mg protein... a 70-fold increase compared to that in untreated vegetative cells. Smaller increases were noted following the inhibition of growth with the drugs cerulenin and vinblastine, as well as after the inhibition of de novo GMP synthesis with the drug mycophenolic acid or the partial inhibition of de novo AMP synthesis with analogs of hadacidin, N-hydroxyglycine and N-formylglycine. In addition, when the activity of two other enzymes involved in purine metabolism, namely adenosine kinase and hypoxanthine-guanine phosphoribosyl transferase, was measured in vegetative cells, and the activity of both compared to that measured in starvation and hadacidin induced growth-arrested cells, showed no significant changes. These data suggest that the changes in the activity of the AMP deaminase are in response to nutrient deprivation and further, that as a consequence of the increase in AMP deaminase activity, ammonia will be produced and an increase in pH should follow. The production of ammonia and its effect on development implicates the AMP deaminase in the early differentiation of this organism.     

Lymphocyte and macrophage adenyl cyclase activity in animals with enhanced cell-mediated resistance to infection and tumors.

As cyclic AMP has been associated with the inhibition of lymphocyte cytotoxicity, studies were performed to investigate adenyl cyclase activity in lymphocytes and macrophages of Toxoplasma-infected mice in which the efferent limb of the cell-mediated immune response had previously been found to be activated. In peritoneal or splenic lymphocytes from $\text{Balb}\text{c}$ mice chronically infected with Toxoplasma in which growth of an isogeneic bladder tumor was found to be inhibited, adenyl cyclase activity was significantly less than in lymphocytes from uninfected control mice. Stimulation by prostaglandin E₁ or NaF in vitro led to higher levels of adenyl cyclase activity in lymphocytes from unifected animals than in cells from Toxoplasma-infected animals. Similar observations were made with peritoneal macrophages from Toxoplasma-infected and uninfected mice. Lower levels of adenyl cyclase activity were also found in lymphocytes from tumor-bearing mice than in lymphocytes from nontumor-bearing controls. These data suggest that production of cyclic AMP by lymphocytes is inhibited with activation of certain cell-mediated immune functions.     

Sensitization of adenylate cyclase: a general mechanism of neuroadaptation to persistent activation of Galpha(i/o)-coupled receptors?

Acute activation of Galphas-coupled receptors stimulates cyclic AMP accumulation leading to the activation of downstream signaling cascades. These Galphas-mediated events can be countered by acute activation of inhibitory G proteins (Galpha(i/o)), which inhibit the activity of adenylate cyclase, thereby attenuating cyclic AMP accumulation. Furthermore, an additional, less direct mechanism for Galpha(i/o) proteins modulation of cyclic AMP signaling also has been described. Persistent activation of several Galpha(i/o)-coupled receptors has been shown to result in a subsequent paradoxical enhancement of adenylate cyclase activity in response to drug-stimulated cyclic AMP accumulation. This sensitization of adenylate cyclase likely represents a cellular adaptive response following prolonged activation of inhibitory receptors. Recent advances in our knowledge of G protein signaling, adenylate cyclase regulation, and other cellular signaling mechanisms have extensively increased our insight into this phenomenon. It is now thought that sensitization occurs as part of a compensatory mechanism by which the cell adapts to chronic inhibitory input. Such a mechanism may be involved in modulating Galphas-coupled receptor signaling following neurotransmitter elevations that occur in psychiatric disease states or following the administration of many drugs of abuse. This review will focus on recent advances in the understanding of molecular signaling pathways that are involved in sensitization and describe the potential role of sensitization in neuronal cell function.     

An interferon-induced increase in cyclic AMP levels precedes the establishment of the antiviral state.

Mouse interferon induces an increase in cyclic AMP levels in interferon-sensitive mouse Ly cells but not in interferon-insensitive human KB-3 cells. The interferon-induced elevation in cyclic AMP precedes the induction of antiviral activity. Although interferon stimulation of the adenylate cyclase activity of Ly cell plasma membranes has not been detected, interferon is effective in stimulating this activity in plasma membranes from rat thyroid cells.     

A maritime pine antimicrobial peptide involved in ammonium nutrition

A large family of small cysteine-rich antimicrobial peptides (AMPs) is involved in the innate defence of plants against pathogens. Recently, it has been shown that AMPs may also play important roles in plant growth and development. In previous work, we have identified a gene of the AMP β-barrelin family that was differentially regulated in the roots of maritime pine (Pinus pinaster Ait.) in response to changes in ammonium nutrition. Here, we present the molecular characterization of two AMP genes, PpAMP1 and PpAMP2, showing different molecular structure and physicochemical properties. PpAMP1 and PpAMP2 displayed different expression patterns in maritime pine seedlings and adult trees. Furthermore, our expression analyses indicate that PpAMP1 is the major form of AMP in the tree, and its relative abundance is regulated by ammonium availability. In contrast, PpAMP2 is expressed at much lower levels and it is not regulated by ammonium. To gain new insights into the function of PpAMP1, we over-expressed the recombinant protein in Escherichia coli and demonstrated that PpAMP1 strongly inhibited yeast growth, indicating that it exhibits antimicrobial activity. We have also found that PpAMP1 alters ammonium uptake, suggesting that it is involved in the regulation of ammonium ion flux into pine roots.