Inhibition of ornithine decarboxylase induction of interferon (alpha + beta) and its reversal by dibutyryl adenosine 3',5'-monophosphate

Interferon (alpha + beta) given to C3H/HeN mice intraperitoneally inhibited increases in the activities of adenylate cyclase and ornithine decarboxylase after partial hepatectomy. The inhibition of ornithine decarboxylase was prevented by administration of dibutyryl cAMP. Core (2'-5')oligo(adenylate), i.e. A2'p5'A2'p5'A or (A2'p)2A, as well as interferon inhibited the increases in these two enzymes caused by partial hepatectomy. The inhibition by (A2'p)2A of ornithine decarboxylase activity was reversed by dibutyryl cAMP. These results suggested that the activity of interferon was similar to that of (A2'p)2A and that the inhibition of ornithine decarboxylase induction caused by these agents resulted from the inhibition of adenylate cyclase activity.     

Implantation in ovariectomized mice treated with dibutyryl adenosine 3',5'-monophosphate (dibutyryl cyclic AMP).

Experiments are described that demonstrate that uterine intraluminal injection of a 1-25 mM-solution of dibutyryl cyclic AMP (dcAMP) in phosphate buffered saline (PBS) induced implantation in ovariectomized pregnant mice. Pretreatment with progesterone was essential for this effect. When PBS was injected alone, it did not induce implantation in mice treated with progesterone. Bilateral adrenalectomy had no effect on the ability of dcAMP to substitute for oestradiol, showing that the effect was not due to dcAMP-induced oestrogen synthesis in the adrenal cortex. It is suggested that the dcAMP may act at the level of the uterus, the embryo, or both.     

Phosphoinositide 3-kinase activity is required for biphasic stimulation of cyclic adenosine 3',5'-monophosphate by relaxin

The G protein-coupled receptors LGR7 and LGR8 have recently been identified as the primary receptors for the polypeptide hormone relaxin and relaxin-like factors. RT-PCR confirmed the existence of mRNA for both LGR7 and LRG8 in THP-1 cells. Whole cell treatment of THP-1 cells with relaxin produced a biphasic time course in cAMP accumulation, where the first peak appeared as early as 1-2 min with a second peak at 10-20 min. Selective inhibitors for phosphoinositide 3-kinase (PI3K), such as wortmannin and LY294002, showed a dose-dependent inhibition of relaxin-mediated increases in cAMP, specific for the second peak of the relaxin time course. Adenylyl cyclase activation by relaxin in purified plasma membranes from THP-1 cells was not inhibited by LY294002, consistent with a mechanism involving direct stimulation by a Galphas-coupled relaxin receptor. However, reconstitution of membranes with cytosol from THP-1 cells enhanced adenylyl cyclase activity and restored LY294002 sensitivity. In addition, relaxin increased PI3K activity in THP-1 cells. Neither the effects of relaxin nor the inhibition of relaxin by LY294002 was mediated by the activity of phosphodiesterases. Taken together, we show that PI3K is required for the biphasic stimulation of cAMP by relaxin in THP-1 cells and present a novel signal transduction pathway for the activation of adenylyl cyclase by a G protein-coupled receptor.     

Comparison of isoproterenol and dibutyryl adenosine cyclic 3',5'-monophosphate stimulation of thyroxine 5'-deiodinase activity in cultured pineal glands from euthyroid and hypothyroid rats

Thyroxine 5'-deiodinase was increased by isoproterenol and dibutyryl adenosine cyclic 3',5'-monophosphate in a dose- and time-related manner in cultured rat pineal gland. Basal and stimulated activity was higher in glands from hypothyroid than from euthyroid animals. Our data suggest direct beta-adrenergic stimulation of intracellular cyclic AMP may be involved in the regulation of pineal thyroxine 5'-deiodinase activity.     

An equilibrium study of the cooperative binding of adenosine cyclic 3',5'-monophosphate and guanosine cyclic 3',5'-monophosphate to the adenosine cyclic 3',5'-monophosphate receptor protein from Escherichia coli

The binding of adenosine cyclic 3',5'-monophosphate (cAMP) and guanosine cyclic 3',5'-monophosphate (cGMP) to the adenosine cyclic 3',5'-monophosphate receptor protein (CRP) from Escherichia coli was investigated by equilibrium dialysis at pH 8.0 and 20 degrees C at different ionic strengths (0.05--0.60 M). Both cAMP and cGMP bind to CRP with a negative cooperativity that is progressively changed to positive as the ionic strength is increased. The binding data were analyzed with an interactive model for two identical sites and site/site interactions with the interaction free energy--RT ln alpha, and the intrinsic binding constant K and cooperativity parameter alpha were computed. Double-label experiments showed that cGMP is strictly competitive with cAMP, and its binding parameters K and alpha are not very different from that for cAMP. Since two binding sites exist for each of the cyclic nucleotides in dimeric CRP and no change in the quaternary structure of the protein is observed on binding the ligands, it is proposed that the cooperativity originates in ligand/ligand interactions. When bound to double-stranded deoxyribonucleic acid (dsDNA), CRP binds cAMP more efficiently, and the cooperativity is positive even in conditions of low ionic strength where it is negative for the free protein. By contrast, cGMP binding properties remained unperturbed in dsDNA-bound CRP. Neither the intrinsic binding constant K nor the cooperativity parameter alpha was found to be very sensitive to changes of pH between 6.0 and 8.0 at 0.2 M ionic strength and 20 degrees C. For these conditions, the intrinsic free energy and entropy of binding of cAMP are delta H degree = -1.7 kcal . mol-1 and delta S degree = 15.6 eu, respectively.     

Specific protein phosphorylation during stimulation of amylase secretion by beta-agonists or dibutyryl adenosine 3',5'-monophosphate in the rat parotid gland

The present study was undertaken in order to examine the possible involvement of protein phosphorylation during beta-adrenergic stimulation in the rat parotid gland. Isolated parotid gland slices were stimulated by either isoproterenol or dibutyryl adenosine 3',5'-monophosphate (Bt2cAMP) in the presence or absence of propranolol. Amylase output was measured as a parameter for the degree of stimulation of secretion. Stimulation of secretion by either isoproterenol or Bt2AMP was associated with phosphorylation of three protein bands as revealed by sodium dodecylsulfate/polyacrylamide gel electrophoresis and autoradiography. The apparent molecular weights of the three proteins were 35,100 (protein I), 25,700 (protein II) and 20,400 (protein III). After cell fractionation by differential and gradient centrifugation, protein I was enriched in a light membrane fraction most likely corresponding to the plasma membrane as revealed by marker enzyme analysis. Proteins II and III were recovered in a denser fraction containing mainly mitochondria and rough microsomes. The effect of isoproterenol but not that of Bt2cAMP on phosphorylation of all three protein bands was completely abolished by propranolol. The different time course in the stimulation of amylase secretion by isoproterenol and Bt2cAMP respectively was reflected by corresponding differences in the time course of protein phosphorylation.