Heterooligomerization between vasotocin and corticotropin-releasing hormone (CRH) receptors augments CRH-stimulated 3',5'-cyclic adenosine monophosphate production

In birds, ACTH release from the anterior pituitary gland during stress is controlled by CRH and arginine vasotocin (AVT). Using 5-wk-old male chicks, simultaneous iv injections of CRH and AVT were found to result in a greater than additive increase in plasma corticosterone levels compared with that obtained with individual administration of either peptide hormone. In order to investigate molecular mechanisms underlying this observation, the chicken CRH receptor (CRHR) and vasotocin VT2 receptor (VT2R) were fused to cyan and yellow fluorescent proteins and expressed in HeLa cells. The resulting CRHR and VT2R fusion proteins were expressed appropriately in the plasma membrane and were found to couple to downstream signal transduction pathways. Quantitative fluorescence resonance energy transfer (FRET) analysis was used to determine whether the CRHR and VT2R formed heterodimers. In the absence of CRH and AVT, the FRET efficiency was 15-18%, and the distance between receptors was 5-6 nm. Treatment of the cells that expressed both cyan fluorescent protein-CRHR and yellow fluorescent protein-VT2R with CRH or AVT alone did not lead to a significant change in the FRET efficiency. However, simultaneous addition of these hormones increased the efficiency of the FRET signal and decreased the distance between the two receptors. In HeLa cells expressing both CRHR and VT2R, treatment with CRH and AVT resulted in a significant increase in cAMP production over that with CRH alone, indicating that heterodimer formation may enhance the ability of the CRHR to activate downstream signal transduction.     

Multihormonal regulation of the human, rat, and bovine growth hormone promoters: differential effects of 3',5'-cyclic adenosine monophosphate, thyroid hormone, and glucocorticoids

We have analyzed the effects of a variety of hormones on activity of the rat GH (rGH), human GH, (hGH), and bovine GH (bGH) promoters. After transient transfection of rat pituitary tumor cells, all three promoters are induced by addition of 8-bromo-cAMP. Sequences required for the cAMP responsiveness of the hGH and rGH promoter lie within 183 base pairs of the mRNA start site. Although the rGH promoter is thyroid hormone (T3) responsive in this system, a construct containing 2.7 kilobases of the hGH promoter 5'-flanking sequences is not. Since we also found that the bGH promoter is T3 responsive in these cells, the hGH results are not likely to be due to a species specific factor required for induction in rat pituitary cells. The hGH promoter is weakly induced by dexamethasone whereas the rGH promoter does not respond to glucocorticoids. The hGH and rGH promoters are not responsive to TRH. These results illustrate the potential heterogeneity in hormonal responses of the same gene in different species.     

Hydrolysis of 2',3'-cyclic adenosine monophosphate and 3',5'-cyclic adenosine monophosphate in subcellular fractions of normal and neoplastic mouse spleen

A comparison has been made between the capacity to hydrolyse 2′,3′-cyclic adenosine monophosphate and 3′,5′-cyclic adenosine monophosphate in subcellular fractions of normal and neoplastic (lymphosarcoma) spleen of C₅₇BL mice. The effect of X-irradiation on these activities was tested. Subcellular fractionation of normal and lymphosarcoma spleen points to a different overall localization of the enzymes. The 2′,3′-cyclic nucleotide phosphodiesterase (2′,3′-cAMPase) has its highest specific activity in the particulate fractions of the cell, while the data on 3′,5′-cyclic nucleotide phosphodiesterase (3′,5′-cAMPase) show the highest activity in the soluble fraction. The 2′,3′-cAMPase activity is higher in the tumor as compared to the normal tissue, while the opposite holds for 3′,5′-cAMPase. Total body irradiation of normal mice with a dose of 600 rads of X-rays, results in a clear drop in 2′,3′-cAMPase 48 hours after the exposure. The 3′,5′-cAMPase is hardly affected at this time. Neither imidazol nor Mg⁺⁺ has any influence on the 2′,3′-cAMPase. The pH optimum for 3′,5′-cAMPase and 2′,3′-cAMPase appears to be 7.7 and 6.2 respectively. This report suggests a no-identity of the two enzymes in mouse spleen, a situation different from that found in certain plants.     

Uptake of adenosine 3',5'-cyclic monophosphate and isoproterenol by filarial parasites

The filarial parasites Litomosoides carinii and Dipetalonema viteae both show transcuticular uptake of adenosine 3',5'-cyclic monophosphate but isoproterenol is taken up by D. viteae only. The importance of this difference is discussed from the point of view of metabolic regulation. Inhibition of uptake by lectins indicates the involvement of surface sugar moieties in the transport processes.     

Regulation of Ig-induced eosinophil degranulation by adenosine 3',5'-cyclic monophosphate.

We have investigated the effects of cAMP on Ig-induced human eosinophil activation. Stimulation of human normodense eosinophils with IgG- or secretory IgA (sIgA)-coated Sepharose beads induced cellular degranulation, as measured by the release of the granule protein, eosinophil-derived neurotoxin (EDN). Pretreatment with cAMP analogs (N6,O2,-dibutyryl adenosine-3,':5' cyclic monophosphate; 8-bromoadenosine 3':5' cyclic monophosphate; or N6-benzoyladenosine 3':5' cyclic monophosphate) or cAMP phosphodiesterase-inhibitors (theophylline or isobutylmethyl xanthine (IBMX] strongly inhibited Ig-induced human eosinophil degranulation. The beta-adrenoceptor agonists, isoproterenol and salbutamol, induced relatively low level increases in intracellular cAMP, and weakly suppressed EDN release induced by IgG-coated beads. However, cellular pretreatment with IBMX synergistically enhanced the inhibitory effects of isoproterenol or salbutamol on both IgG and sIgA-induced eosinophil degranulation. Similarly, PGE2 treatment increased intracellular cAMP concentrations in eosinophils and correspondingly inhibited the Ig-dependent cellular degranulation response: co-incubation with IBMX further enhanced both effects of PGE2. Finally, cholera toxin, which irreversibly activates the stimulatory guanine nucleotide-binding protein linked to adenylyl cyclase, strongly inhibited the release of EDN from IgG- or sIgA-stimulated eosinophils. The time-dependent accumulation of cAMP in cholera toxin-treated cells closely paralleled the time courses of inhibition of IgG- and sIgA-induced EDN release after toxin exposure. These data indicate that the cAMP-dependent signal transduction mechanism in eosinophils exerts a negative modulatory effect on the cellular degranulation responses induced by sIgA or IgG. The inhibitory effects of cAMP on eosinophil activation may provide an important physiologic and a clinically relevant therapeutic mechanism for limiting the release of eosinophil-derived cytotoxic proteins during certain allergic or inflammatory responses in vivo.     

Circadian variations in plasma 3':5'-cyclic adenosine monophosphate and 3':5'-cyclic guanosine monophosphate of normal adults.

Circadian variations in plasma cyclic AMP and cyclic GMP were studied in thirteen male subjects (20–22 years old) under controlled invironmental condition. Plasma collections were made every six hours. Cyclic AMP and cyclic GMP were determined by radioimmunoassay. Individual values of plasma cyclic AMP at 0800 are between 13.0 and 25.8 pmole/ml, and cyclic GMP between 2.5 and 7.0 pmole/ml. Cyclic AMP demonstrated the circadian variation with the maximum level at 1400 and the minimum at 0200, and cyclic GMP with the highest level at 1400 and the lowest level at 0800.     

Identification and characterization of a 3',5'-cyclic adenosine monophosphate-responsive element in the human corticotropin-releasing hormone gene promoter.

The regulation of human corticotropin-releasing hormone (hCRH) gene promoter activity by inducers of cAMP was investigated by transient transfection with a construct containing the hCRH gene promoter fused to the chloramphenicol acetyltransferase gene. Expression of hCRH-chloramphenicol acetyltransferase was strongly enhanced by forskolin in the neuroblastoma SK-N-MC and choriocarcinoma JAR cell lines. Overexpression of the catalytic subunit of protein kinase A dispensed the need for forskolin, and cotransfection of cAMP-responsive element-binding protein cDNAs enhanced forskolin-dependent expression of the hCRH promoter. Progressive 5'-end deletions of the hCRH promoter delineated a cAMP- responsive region between -226 and -164 base pairs. This fragment contained the sequence TGACGTCA at -221 base pairs, consistent with the consensus motif for a CRE. A homologous oligonucleotide responded to cAMP when cloned in either orientation in front of the thymidine kinase promoter. However, the level of constitutive and inductive cAMP expression was dependent on the cell line and on intrinsic properties of the promoter. Mutation of the wild type CRH-CRE sequence into an AP-1 site (TGAGTCA) completely abolished stimulation by cAMP. In contrast, coexpression of the catalytic subunit of protein kinase A dispensed the need for stimulation with forskolin, which showed that the CRH-CRE oligonucleotide served as a functional equivalent of the native CRE element.     

Effects of adenosine 5'-monophosphate and adenosine 3',5'-cyclic monophosphate on l cells.

The adenine nucleotides, 5'-AMP and 3',5'-cyclic AMP block L cells in the S-phase of the cell cycle. The intracellular level of cyclic AMP is reduced after incubation of cells with 5'-AMP, and rates of uridine transport are increased after incubation with either 5'-AMP or cyclic AMP. On the contrary, cyclic AMP levels are increased and uridine transport decreased in cells treated with an inhibitor of the cyclic AMP phosphodiesterase. This inhibitor partially reverses the growth-inhibitory effect of cyclic AMP, indicating that a breakdown product is the effective inhibitor of growth. The inhibition of cell growth induced by the adenine nucleotides is prevented by uridine, suggesting that the block in S is due to a lack of availability of pyrimidines.     

The regulation of adenosine 3':5'-cyclic monophosphate accumulation in glia by alpha-adrenergic agonists.

This study examines the α-adrenergic modulation of cAMP accumulation in glia. This phenomenon occurs in three different glial culture systems: (a) primary surface cultures of glia, (b) reaggregate cultures containing neurons and glia, and (c) C6 glioma cells. The turn-on and turn-off times of α-adrenergic modulation appear to be in the same time frame as that of agents which normally increase cAMP accumulation. However, even after prolonged treatment periods, refractoriness does not develop to the modulating capacity of α-adrenergic agents. Clonidine, reported to act as both an α-adrenergic agonist and antagonist in the central nervous system, was found to interact with glia as an α-adrenergic agonist. Alpha- adrenergic modulation was not diminished by the addition of ethylene- glycol-bis-(β-amino-ethyl ether)N, N′-tetra-acetic acid (EGTA) to culture media, suggesting that external calcium is not required for this effect. These results illustrate the complexity of glial pharmacology and need for more defined systems which allow the examination of characterized populations of brain cells.     

Differences and similarities between guanosine 3',5'-cyclic monophosphate phosphodiesterase and adenosine 3',5'-cyclic monophosphate phosphodiesterase activities in neuroblastoma cells in culture.

There are phosphodiesterase activities in both particulate and supernatant fractions which hydrolyze guanosine 3',5'-cyclic monophosphate (cGMP) and adenosine 3',5'-cyclic monophosphate (cAMP) with an apparent Km of 2-8 muM and with an apparent Km of 44-222 muM. 4-(3-Butoxy-4-methoxybenzyl-2-imidazolidinone (RO20-1724) did not inhibit cGMP phosphodiesterase activity in homogenates of mouse neuroblastoma cells, but markedly inhibited cAMP phosphodiesterase activity. Papaverine and theophylline inhibited both cGMP and cAMP phosphodiesterase activities to about the same extent. The former was more potent than the latter. The specific activity of cGMP phosphodiesterase as a function of protein concentrations first increased and then decreased. The specific activity of cAMP phosphodiesterase decreased under a similar experimental condition.     

Stimulation of galactokinase synthesis in Escherichia coli by adenosine 3',5'-cyclic monophosphate

Adenosine 3',5'-cyclic monophosphate (cyAMP) stimulates the rate of synthesis of galactokinase in glycerol-grown Escherichia coli both when production of the enzyme is induced by d-fucose and when it is repressed by glucose in the presence of inducer. cyAMP also stimulates the synthesis of galactokinase in constitutive strains B78A (R(-)) and R10 (O(c)), and overcomes the transient repression of galactokinase synthesis caused by glucose.