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.     

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.     

Ketotifen increases cyclic adenosine 3',5'-monophosphate in intact human lymphocyte and potentiates other adenylate cyclase activating agents

We have investigated the effects of ketotifen on the cyclic adenosine 3',5'-monophosphate (cyclic AMP) response of intact human lymphocyte and its interaction with adenylate cyclase activating agents. In the presence of cyclic AMP phosphodiesterase inhibitor (3-isobutyl-1-methyl-xanthine), ketotifen (10(-8)-10(-4) M) caused an 80% increase in cyclic AMP content of human lymphocyte, a magnitude similar to that observed with hydrocortisone. The cyclic AMP level peaked at about 15 minutes and remained elevated for at least 45 minutes. In addition, ketotifen (10(-6)-10(-4) M) markedly potentiated the effect of several adenylate cyclase stimulating agents, including L-isoproterenol, prostaglandin E1 and forskolin. The biochemical mechanisms underlying these effects are unknown. It may be at least partly related to the ability of ketotifen to reverse and prevent beta 2 adrenoceptor desensitization and to promote the formation of hormone - nucleotide - high affinity receptor complex. These effects may contribute to its prophylactic effect in the treatment of bronchial asthma.     

Intracellular localization of phosphodiesterase induced by cyclic adenosine 3',5'-monophosphate in Dictyostelium discoideum

The intracellular distribution of phosphodiesterase [EC 3.1.4.17] induced by cyclic adenosine 3',5'-monophosphate (cAMP) in Dictyostelium discoideum was studied. When cAMP-treated cells were homogenized and fractionated according to the method of de Duve et al. ((1955) Biochem, J. 60, 604), the specific activity of phosphodiesterase was highest in the light mitochondrial fraction. Peaks of specific activities of alkaline phosphatase (marker enzyme of membrane) and catalase (marker enzyme of peroxisomes) also appeared in the same fraction as phosphodiesterase. However, after centrifugation of the light mitochondrial fraction in a sucrose density gradient, the activity of phosphodiesterase was clearly separated with that of catalase (density 1.19 g/ml) and showed three peaks at lower density (1.10, 1.13, 1.17 g/ml) with good reproducibility. Some parts (1.13, 1.17 g/ml) of the activity in the gradient overlapped with alkaline phosphatase activity, but in the density fraction of 1.10 g/ml the activity of alkaline phosphatase was hardly detectable. When the light mitochondrial fraction was treated with Emulgen 108, or sonicated, phosphodiesterase was more easily solubilized than alkaline phosphatase and catalase, and was found in supernate after centrifugation at 20,000 X g for 30 min. In order to distinguish the locations of the three enzymes, the supernatant of the light mitochondrial fraction treated with Emulgen 108 was subjected to charge shift electrophoresis. The electrophoretic mobilities of phosphodiesterase and catalase were unaffected by ionic detergent. However, alkaline phosphatase shifted towards the anode in the presence of anionic detergent (sodium deoxycholate), and shifted towards the cathode in cationic detergent (cetyltrimethylammonium bromide), relative to nonionic detergent (Emulgen 108) alone. Thus, some part of the phosphodiesterase induced by cAMP may be associated with the plasma membrane, but the remainder is localized in some kind of intracellular particle of lower density. Moreover, the association with the membrane or particle is more easily dissociated than that of alkaline phosphatase, and the liberated phosphodiesterase is rather hydrophilic.     

Compartmentation of cyclic adenosine 3',5'-monophosphate signaling in caveolae.

The cAMP-signaling pathway is composed of multiple components ranging from receptors, G proteins, and adenylyl cyclase to protein kinase A. A common view of the molecular interaction between them is that these molecules are disseminated on the plasma lipid membrane and random collide with each other to transmit signals. A limitation to this idea, however, is that a signaling cascade involving multiple components may not occur rapidly. Caveolae and their principal component, caveolin, have been implicated in transmembrane signaling, particularly in G protein-coupled signaling. We examined whether caveolin interacts with adenylyl cyclase, the membrane-bound enzyme that catalyzes the conversion of ATP to cAMP. When overexpressed in insect cells, types III, IV, and V adenylyl cyclase were localized in caveolin-enriched membrane fractions. Caveolin was coimmunoprecipitated with adenylyl cyclase in tissue homogenates and copurified with a polyhistidine-tagged form of adenylyl cyclase by Ninitrilotriacetic acid resin chromatography in insect cells, suggesting the colocalization of adenylyl cyclase and caveolin in the same microdomain. Further, the regulatory subunit of protein kinase A (RIIalpha, but not RIalpha) was also enriched in the same fraction as caveolin. Gsalpha was found in both caveolin-enriched and non-caveolin-enriched membrane fractions. Our data suggest that the cAMP-signaling cascade occurs within a restricted microdomain of the plasma membrane in a highly organized manner.     

The effects of follicle-stimulating hormone and cyclic guanosine 3',5'-monophosphate on cyclic adenosine 3',5'-monophosphate-phosphodiesterase and resumption of meiosis in hamster cumulus-oocyte complexes

The effects of follicle-stimulating hormone (FSH) and cyclic guanosine 3',5'-monophosphate (cGMP) on spontaneous oocyte maturation and cyclic adenosine 3',5'-cumulus-monophosphate phosphodiesterase activity (cAMP-PDE) were evaluated by using cumulus-oocyte complexes (COCs) from proestrous hamsters. After a 2-h incubation period, FSH (10 micrograms/ml and 1 microgram/ml) reduced the percentage of maturing oocytes compared with controls. This inhibition was partially overcome when cGMP-elevating agents (8-Bromo-cGMP, atrial natriuretic factor or sodium nitroprusside) were included with FSH. After a 3-h period, incubation with FSH and cGMP-elevating agents alone increased the maturation rate above that of the controls. The accelerating effects of cGMP on the maturation rate appear to be caused by its capacity to lower cAMP levels. Combining FSH (1 microgram/ml) with sodium nitroprusside reduced cAMP levels in COCs (not oocytes) compared with groups exposed to FSH alone. FSH increased cGMP levels in COCs in a dose- and time-dependent manner. Both FSH and cGMP-elevating agents produced a dose-dependent increased cAMP-PDE activity in COCs (not oocytes) following a 2-h incubation period. Together, these results suggest that, in vivo, FSH stimulates a rise in both cAMP and cGMP in COCs. While the increase in cAMP may be the initial meiotic trigger, cGMP may serve to subsequently lower cAMP by activating cAMP-PDE and thus permit the maturational process to continue.     

The effect of relaxin on cyclic adenosine 3',5'-monophosphate concentrations in human endometrial glandular epithelial cells

The effects of relaxin (RLX), forskolin (Fk), and 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro, a phophodeisterase inhibitor) on the accumulation of cyclic adenosine 3',5'-monophosphate (cAMP) in human endometrial glandular epithelial cells were studied. Epithelial glands were isolated from the endometrium by digesting the viable tissue fragments with collagenase. The epithelial glands were incubated with Ro, RLX, and Fk separately or in combination. The amount of cAMP was determined at the end of incubation. A moderate increase in cAMP content was observed in epithelial glands incubated with Ro alone. Accumulation of cAMP after incubation with RLX was observed only in the presence of Ro. Increase of cAMP content in response to RLX and Ro was time- and dose-dependent. The accumulation of cAMP was apparent in 5 min, reached the maximum after 15 min, and remained elevated for 17 h incubation. One nanogram per millileter RLX was effective to increase the cAMP content, with a maximal response at 100 ng/ml. The effect of Ro and the combined effect of Ro and RLX on cAMP accumulation were studied in epithelial glands of 20 endometrial specimens obtained during different stages of the menstrual cycle. When epithelial glands were incubated with Ro alone, the cAMP concentration in glands from proliferative endometria was 120 +/- 67 pmol/mg protein (n = 6, means +/- SD), significantly higher than that of secretory endometria, 42 +/- 37 (n = 14, p = 0.007). RLX and Ro caused an additional increase of cAMP accumulation, 2- to greater than 10-fold increase over the sample incubated with Ro alone. There was no significant difference between proliferative and secretory phases (500 +/- 410, n = 6, and 470 +/- 300, n = 14, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of Escherichia coli growth by cyclic adenosine 3', 5'-monophosphate

Cyclic AMP inhibits growth rate of E. coli Hfr 3000. Doubling times in glucose minimal medium increased from 60 to about 90 minutes with the addition of 5 mM cAMP. This effect is specific since it was not observed when the cyclic nucleotide was replaced by 5′ AMP, ADP, ATP or adenosine. Half maximal inhibition was obtained with 1 to 3 mM cyclic AMP. This inhibition occurs only with those carbon sources which are known to decrease intracellular cyclic AMP levels, i.e. glucose and pyruvate. No inhibition was observed with succinate, malate or glycerol.     

Alpha adrenergic stimulation reduces cyclic adenosine 3',5'-monophosphate generation in rabbit myometrium by two mechanisms

Rabbit myometrium contains postsynaptic alpha-1, alpha-2, and beta-2 adrenoreceptors. The response to endogenous catecholamines depends on the summation of interactions at these receptors and is influenced by the hormonal environment. Estrogen treatment of ovariectomized rabbits increases the alpha adrenergic contractile response whereas progesterone treatment of estrogen primed animals results in a predominance of the beta adrenergic response, which is inhibition of contractions. Of the receptor subtypes, only the alpha-2 receptor concentration is increased at physiological estrogen concentrations. However, alpha-2 receptors have not been shown to be directly involved in myometrial contraction, which appears to be mediated solely by alpha-1 adrenergic interactions. To test whether alpha-2 receptors might indirectly affect contraction by opposing interactions at the beta receptor, we examined the ability of alpha adrenergic stimulation to reduce myometrial cyclic adenosine 3',5'-monophosphate (cAMP) generation. We find that alpha-2 receptors inhibit myometrial ade adenylate cyclase through the guanine nucleotide regulatory protein, Gi. In addition, we find that activation of alpha-1 receptors also reduces cAMP generation. This interaction, which can be demonstrated in the absence but not the presence of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, does not appear to be mediated through Gi. These findings illustrate the complexity of adrenergic interactions in tissues containing several adrenergic subtypes.