Two novel stimuli of cyclic adenosine 3',5'-monophosphate (cAMP) in human lymphocytes.

Polystyrene latex particles (PLP) and zymosan particles (ZP), two commonly employed phagocytic stimuli, were noted to bind to purified human peripheral blood lymphocytes. This interaction was not accompained by ingestion but did lead to a marked increase in intracellular cyclic AMP. The cAMP response to PLP was proportional to the particle cell ratio which, in turn, correlated with the number of membrane-associated particles. After the addition of PLP to lymphocytes, the cAMP response occurred within 2 min, peaked between 4 and 15 min, and returned to baseline by 30 to 60 min. The cAMP response to ZP was similar in onset and duration to that seen with PLP but was less marked (2- to 4-fold vs 25- to 50-fold) and more variable in magnitude. This is probably a reflection of the smaller number of cells interacting with ZP. At high PLP to cell ratios almost all of the lymphocytes bound PLP but only 10 to 28% of the mixed lymphocyte population bound ZP. Two lines of evidence established conclusively that the cAMP response was taking place in the lymphocytes themselves rather than in contaminating cells. 1) When lymphocytes were purified additionally by filtration through a nylon wool column (99 to 100% lymphocytes), they were found to undergo a similar cAMP response to PLP. Since the nylon filtration procedure also removes almost all of the B cells, this further indicates that T cells are capable of undergoing the response. 2) Immunofluorescence studies with anti-cAMP antibody revealed an increase in intralymphocytic cAMP which was primarily adjacent to the site of PLP or ZP attachment. The likely explanation of this data is that PLP and ZP perturb the lymphocyte surface leading to regional activation of membrane-bound adenylate cyclase and subsequent cAMP accumulation. Although the physiologic significance of these observations remains to be determined, the results: 1) provide histologic confirmation for the concept of cAMP compartmentablization, 2) clarify conflicting results regarding the localization of cAMP accumulation during the phagocytosis of PLP by mixed leukocyte populations, and 3) suggest that this experimental system may allow an analysis of the mechanism by which perturbations of the lymphocyte surface modulate cAMP.     

Specificity of the cyclic adenosine 3',5'-monophosphate signal in granulosa cell function

Cyclic AMP signaling is involved in most aspects of differentiation and maturation of the granulosa cells in the ovarian follicle. As the genetic programs activated at different stages of follicle growth maturation are being elucidated, it is becoming increasingly difficult to reconcile the simplicity of the cAMP cascade with the complexity and the divergent patterns of gene expression activated in these cells. To account for these divergent outcomes of the cAMP signal, three aspects of this signaling cascade in granulosa cells will be reviewed. We will discuss the evidence for gonadotropin receptors coupling to different G proteins and effectors. Next, we will explore the possibility that the temporal and spatial dimensions of the cAMP signal itself may contribute to the diverse outcomes. Finally, we will summarize available data showing that the cAMP signal is distributed through several cascades of kinase activation. It is hoped this compendium will provide a framework with which to understand how the initial signals activated by gonadotropins control the complex patterns of gene expression that are required for follicle maturation and ovulation.     

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.     

Urinary adenosine 3',5'-monophosphate (cyclic AMP) in normal and cryptorchid boys.

The 24-hour urinary excretion of cyclic AMP was determined in 102 normal boys aged 1.9-16.9 years and in 136 cryptorchids aged 2.5-16.9 years. A marked increase of the normal cyclic AMP excretion was found in pubertal years. There was a positive correlation between urinary excretion of cyclic AMP and the excretion of testosterone, androstenedione, LH and FSH. A positive correlation was also found between cyclic AMP excretion and height and weight, respectively. Mean cyclic AMP excretion of bilateral and unilateral cryptorchids was normal in all bone age groups except in unilateral cases with bone age 8-9.9 years and bone aged greater than or equal to 14 years. In these two groups, mean cyclic AMP excretion was moderately increased. After HCG stimulation of 25 cryptorchids, urinary cyclic AMP excretion varied between increased, unchanged and decreased values. The cyclic AMP excretion changes observed in some of our patients were difficult to interpret and were possibly of unspecific nature. Further information about the testiclar cyclic AMP secretion and the relationship between this nucleotide and sexual hormones may be obtained from studies in testicular biopsy tissue.     

Biphasic effect of calcium on luteinizing hormone-stimulated cyclic adenosine 3',5'-monophosphate production in granulosa cells of the fowl (Gallus domesticus).

Both cAMP and Ca2+ play important roles in the steroidogenic action of LH in hen granulosa cells. However, the interaction of these intracellular messengers is not fully understood. In the present study we used two calcium ionophores (ionomycin and A23187), as well as trifluoperazine (TFP), an inhibitor of calmodulin, to investigate LH- and forskolin-induced cAMP production in granulosa cells isolated from the largest (F1) preovulatory follicle of White Leghorn laying hens. Between 0.1 and 1.0 microM, both ionophores significantly potentiated cAMP responses to LH in the presence of 0.1 mM extracellular Ca2+. When calcium was omitted from the medium, ionophores had no effect. When either calcium was raised above 1 mM, or the concentration of ionophores was increased above 1 microM, LH-induced cAMP production was drastically inhibited. In the presence of 0.5-2.0 mM calcium, A23187 inhibited forskolin-promoted cAMP synthesis. TFP, while having no effect on basal cAMP, suppressed LH-induced responses and the potentiating effect of ionomycin. It is concluded that for full activation of the adenylate cyclase/cAMP system by LH, Ca-calmodulin is required at a site upstream from the catalytic component of the enzyme. However, high intracellular Ca2+ and/or other effects of ionophores (such as uncoupling of oxidative phosphorylation) inhibit LH-induced cAMP production.     

Crystal structures of RIalpha subunit of cyclic adenosine 5'-monophosphate (cAMP)-dependent protein kinase complexed with (Rp)-adenosine 3',5'-cyclic monophosphothioate and (Sp)-adenosine 3',5'-cyclic monophosphothioate, the phosphothioate analogues of cAMP

Cyclic adenosine 5'-monophosphate (cAMP) is an ancient signaling molecule, and in vertebrates, a primary target for cAMP is cAMP-dependent protein kinase (PKA). (R(p))-adenosine 3',5'-cyclic monophosphothioate ((R(p))-cAMPS) and its analogues are the only known competitive inhibitors and antagonists for cAMP activation of PKA, while (S(p))-adenosine 3',5'-cyclic monophosphothioate ((S(p))-cAMPS) functions as an agonist. The crystal structures of a Delta(1-91) deletion mutant of the RIalpha regulatory subunit of PKA bound to (R(p))-cAMPS and (S(p))-cAMPS were determined at 2.4 and 2.3 A resolution, respectively. While the structures are similar to each other and to the crystal structure of RIalpha bound to cAMP, differences in the dynamical properties of the protein when (R(p))-cAMPS is bound are apparent. The structures highlight the critical importance of the exocyclic oxygen's interaction with the invariant arginine in the phosphate binding cassette (PBC) and the importance of this interaction for the dynamical properties of the interactions that radiate out from the PBC. The conformations of the phosphate binding cassettes containing two invariant arginine residues (Arg209 on domain A, and Arg333 on domain B) are somewhat different due to the sulfur interacting with this arginine. Furthermore, the B-site ligand together with the entire domain B show significant differences in their overall dynamic properties in the crystal structure of Delta(1-91) RIalpha complexed with (R(p))-cAMPS phosphothioate analogue ((R(p))-RIalpha) compared to the cAMP- and (S(p))-cAMPS-bound type I and II regulatory subunits, based on the temperature factors. In all structures, two structural solvent molecules exist within the A-site ligand binding pocket; both mediate water-bridged interactions between the ligand and the protein. No structured waters are in the B-site pocket. Owing to the higher resolution data, the N-terminal segment (109-117) of the RIalpha subunit can also be traced. This strand forms an intermolecular antiparallel beta-sheet with the same strand in an adjacent molecule and implies that the RIalpha subunit can form a weak homodimer even in the absence of its dimerization domain.     

Photoaffinity labeling of the adenosine cyclic 3',5'-monophosphate receptor protein of Escherichia coli with 8-azidoadenosine 3',5'-monophosphate

Photoaffinity labeling of the cAMP receptor protein (CRP) of Escherichia coli with 8-azidoadenosine 3',5'-monophosphate (8-N3cAMP) has been demonstrated. 8-N3cAMP is able to support the binding of (3H)d(I-C)n by CRP, indicating that it is a functional cAMP analogue. Following irradiation at 254 nm, (32P)-8-N3cAMP is photocross-linked to CRP. Photolabeling of CRP by (32P)-8-N3cAMP is inhibited by cAMP but not by 5'AMP. The data indicate that (32P)-8-N3cAMP is covalently incorporated following binding at the cAMP binding site of CRP. The (32P)-8-N3cAMP-CRP digested with chymotrypsin was analyzed by NaDodSO4-polyacrylamide gel electrophoresis. Of the incorporated label, one-third remains associated with the amino-proximal alpha core region of CRP [Eilen, E., Pampeno, C., & Krakow, J.S. (1978) Biochemistry 17, 2469] which contains the cAMP binding domain; the remaining two-thirds of the label associated with the beta region are digested. Limited proteolysis of the (32P)-8-N3cAMP-CRP by chymotrypsin in the presence of NaDodSO4 shows the radioactivity to be distributed between the molecular weight 9500 (amino-proximal) and 13,000 (carboxyl-proximal) fragments produced. These results suggest that a part of the carboxyl-proximal region is folded over and close enough to the cAMP binding site to be cross-linked by the photoactivated (32P)-8-N3cAMP bound at the cAMP binding site.