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.     

Characterization of a specific adenosine receptor on human lymphocytes.

We have examined the mechanism of action of adenosine, a naturally occurring nucleoside that has profound effects on lymphocyte function. Adenosine (0.01 micrometer to 10 micrometer) increased lymphocytes cAMP levels in a dose-dependent fashion with a maximal (10 micrometer) increase of about 4-fold, whereas adenine, guanosine, and inosine had no effect on lymphocyte cAMP levels at concentrations of 100 micrometer. Adenosine appears to act on the cell surface since 1) 2-chloroadenosine, a poorly metabolized adenosine analogue, was as active as adenosine and 2) dipyridamole, which markedly inhibited [3H]-adenosine uptake by human lymphocytes, did not affect adenosine-induced accumulation of cAMP. The specificity of the adenosine effect was established by showing that the methylxanthine derivatives, theophylline and 3-isobutyl-1-methylxanthine (IBMX), specifically block the accumulation of cAMP in lymphocytes induced by adenosine. Theophylline is a competitive inhibitor of the effect of adenosine, with an estimated dissociation constant of theophylline-receptor complex of about 6.3 X 10(-7) M. The results suggest that adenosine increases the intracellular cAMP content of lymphocytes as a result of its interaction with a specific membrane receptor which results in the activation of adenylate cyclase.     

Trypsin-induced increase in intracellular cyclic AMP of lymphocytes.

Trypsin increases intracellular levels of cylic AMP (cAMP) in lymphocytes. The trypsin-induced increase in cAMP is blocked by specific trypsin inhibitors and by high concentrations of different proteins. Several proteolytic enzymes from various sources, including other pancreatic proteases, do not cause an increase in cAMP under the same experimental conditions. Immobilized trypsin induced the same increase in cAMP as does free trypsin. The trypsin-induced rise in cAMP is not due to inhibition of cAMP phosphodiesterase, but consistent activation of adenylate cyclase by trypsin could not be demonstrated. The extent of the trypsin-induced increase in intracellular cAMP correlates with the type of the lymphocyte and with the state of maturity attained by the cells. Transformed lymphocytes and nonlymphoid cells do not react at all.     

Differential Association of Phosphodiesterase 4D Isoforms with ��2-Adrenoceptor in Cardiac Myocytes

cAMP and protein kinase A (PKA) activation represents a key signaling mechanism upon β-adrenergic stimulation under stress. Both β₁- and β₂-adrenoreceptor (ARs) subtypes induce cAMP accumulation, yet play distinct roles in cardiac contraction and myocyte apoptosis. Differences in controlling cAMP/PKA activities through the assembly of complexes between the receptors and cAMP-specific phosphodiesterases contribute to the distinct biological outcomes. Here, we demonstrate that β₂ARs form signaling complexes with a set of PDE4D isoforms expressed in cardiac myocytes. PDE4D9 and PDE4D8 bind to the β₂AR at resting conditions; however, agonist stimulation induces dissociation of PDE4D9 from the receptor but recruitment of PDE4D8 to the receptor. Agonist stimulation also induces recruitment of PDE4D5 to the β₂AR. Moreover, the receptor-associated PDE4D isoforms play distinct roles in controlling cAMP activities and regulating the PKA phosphorylation of the receptor and myocyte contraction rate responses. Knockdown of PDE4D9 with short hairpin RNA enhances the β₂AR-induced cAMP signaling, whereas knockdown of PDE4D8 only slightly prolongs the receptor-induced cAMP signaling in myocytes. Inhibition of PDE4D9 and PDE4D5 enhances the base-line levels of contraction rates, whereas inhibition of PDE4D9 and PDE4D8 enhances the maximal contraction rate increases upon activation of β₂AR. Our data underscore the complex regulation of intracellular cAMP by β₂AR-associated phosphodiesterase enzymes to enforce the specificity of the receptor signaling for physiological responses.     

Cyclic GMP and lectin-induced lymphocyte activation.

Purified human peripheral lymphocytes incubated with the mitogenic plant lectins phytohemagglutinin and concanavalin A were examined for alterations in intracellular cGMP and cAMP under a variety of experimental conditions and using multiple techniques for the isolation and purification of cGMP and cAMP before assay of the cyclic nucleotides by radioimmunoassay. In contrast to work reported by others, we have been unable to demonstrate consistent increases in cGMP under any of the experimental conditions used and with any of the various purification schemes. In these same experiments exogenous cGMP added to the lymphocytes could be measured, and the immunoreactive material was destroyed by cyclic nucleotide phosphodiesterase, indicating that our inability to measure increases in cGMP was not caused by our inability to measure cGMP. Under identical experimental conditions, small but consistent and statistically significant increases in cAMP were noted. In addition, other parameters of lymphocyte activation, 45Ca uptake (an early parameter), and incorporation of 3H thymidine into DNA were unimpaired. These data call to question the concept of cGMP as the second messenger in lectin-stimulated human peripheral lymphocytes.     

The requirement for cell surface galactosyl residues during oxidative activation of normal and chronic lymphocytic leukemia lymphocytes.

Galactose oxidase stimulated normal and leukemic lymphocytes to undergo DNA synthesis and cell division. Although the response of normal lymphocytes to galactose oxidase was enhanced with neuraminidase pretreatment, substantial activation of leukemic lymphocytes required pretreatment with neuraminidase. Leukemic lymphocytes exhibited maximal response to neuraminidase-galactose oxidase later than that observed in normal lymphocytes. Treatment of lymphocytes with trypsin diminished their response to galactose oxidase. When lymphocytes were pretreated with β-galactosidase to specifically remove cell surface galactosyl residues, the response to galactose oxidase was prevented. The response of normal and leukemic lymphocytes to sodium periodate was also reduced after treatment with galactose oxidase. These data support the concept that oxidation of cell surface galactosyl residues is critical during lymphocyte activation.     

A sodium requirement for mitogen-induced proliferation in human peripheral blood lymphocytes

Mitogen-stimulated DNA synthesis in human peripheral blood lymphocytes is dependent on extracellular Na. DNA synthesis was similarly inhibited in:

1. 1. Cells that were suspended in hypotonic media containing decreased extracellular Na.

2. 2. Cells that were suspended in media containing decreased Na and equimolar replacement with choline.

3. 3. Cells that were suspended in media containing decreased Na and equiosmolar replacement with mannitol.

A decreased PHA-induced DNA synthesis was observed at day 3 even when lymphocytes were exposed to low Na for only the first 3 h and then returned to normal levels of Na. Our studies of protein synthesis indicate that the effect of lowered extracellular Na on DNA synthesis and cell division is not due to an initial inhibition of overall protein synthesis. These data suggest that reduced external Na has a significant effect on some specific early event(s) (3 h) in lymphocyte mitogenesis.     

Effect of cAMP on ciliary function in rabbit tracheal epithelial cells

To study the effect of adenosine 3',5'-cyclic monophosphate (cAMP) on respiratory ciliary activity, we measured ciliary beat frequency (CBF) of rabbit tracheal epithelium by a photoelectric method in response to cAMP analogues and agents that can increase endogenous cAMP production. Addition of 8-bromo-cAMP dose dependently enhanced CBF, with the maximal increase and the concentration necessary to produce a half-maximal response (KD) being 31.0 +/- 3.4% (SE) (P less than 0.001) and 3.2 +/- 1.5 x 10(-7) M, respectively. Other structurally dissimilar cAMP analogues dibutyryl cAMP and chlorophenylthio-cAMP likewise caused increases in CBF. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine and the adenylate cyclase stimulator forskolin also augmented CBF in a dose-dependent fashion and were accompanied by the increases in intracellular concentrations of cAMP. Ciliary discoordination was not observed in any of the experiments. These results suggest that cAMP may accelerate mucociliary clearance through the activation of ciliary motility and that intracellular cAMP levels appear to be an important determinant for the lung mucociliary transport functions.     

Microfilaments and cellular signal transduction: effect of cytochalasin D on the production of cAMP, inositol phosphates, and on calcium movements in rat parotid glands

In rat parotid glands, the involvement of the microfilament system in the cellular signal transmission mechanism was tested by measuring the effect of cytochalasin D (which disturbs the microfilament system) on the production of intracellular second messengers. Cytochalasin D (CD) did not affect unstimulated calcium movements (measured by the 45Ca efflux technique) or inositol phosphate production or cAMP accumulation. Neither did it modify the generation of intracellular second messengers induced by activation of the cholinergic muscarinic receptor (calcium and inositol phosphates). CD dit not affect the cAMP accumulation induced by the activation of the beta-adrenergic receptor whereas it strongly inhibited the calcium movements induced by activation of the same receptor. These data suggest that, in rat parotid glands, calcium movements, induced by beta-adrenergic receptor stimulation need an intact microfilament system to occur, whereas the muscarinic pathway (via IP3) does not.     

Kinesin-II is required for flagellar sensory transduction during fertilization in Chlamydomonas

The assembly and maintenance of eucaryotic flagella and cilia depend on the microtubule motor, kinesin-II. This plus end-directed motor carries intraflagellar transport particles from the base to the tip of the organelle, where structural components of the axoneme are assembled. Here we test the idea that kinesin-II also is essential for signal transduction. When mating-type plus (mt+) and mating-type minus (mt-) gametes of the unicellular green alga Chlamydomonas are mixed together, binding interactions between mt+ and mt- flagellar adhesion molecules, the agglutinins, initiate a signaling pathway that leads to increases in intracellular cAMP, gamete activation, and zygote formation. A critical question in Chlamydomonas fertilization has been how agglutinin interactions are coupled to increases in intracellular cAMP. Recently, fla10 gametes with a temperature-sensitive defect in FLA10 kinesin-II were found to not form zygotes at the restrictive temperature (32 degrees C). We found that, although the rates and extents of flagellar adhesion in fla10 gametes at 32 degrees C are indistinguishable from wild-type gametes, the cells do not undergo gamete activation. On the other hand, fla10 gametes at 32 degrees C regulated agglutinin location and underwent gamete fusion when the cells were incubated in dibutyryl cAMP, indicating that their capacity to respond to the cAMP signal was intact. We show that the cellular defect in the fla10 gametes at 32 degrees C is a failure to undergo increases in cAMP during flagella adhesion. Thus, in addition to being essential for assembly and maintenance of the structural components of flagella, kinesin-II/intraflagellar transport plays a role in sensory transduction in these organelles.     

Mst1 controls lymphocyte trafficking and interstitial motility within lymph nodes

The regulation of lymphocyte adhesion and migration plays crucial roles in lymphocyte trafficking during immunosurveillance. However, our understanding of the intracellular signalling that regulates these processes is still limited. Here, we show that the Ste20-like kinase Mst1 plays crucial roles in lymphocyte trafficking in vivo. Mst1^−/− lymphocytes exhibited an impairment of firm adhesion to high endothelial venules, resulting in an inefficient homing capacity. In vitro lymphocyte adhesion cascade assays under physiological shear flow revealed that the stopping time of Mst1^−/− lymphocytes on endothelium was markedly reduced, whereas their L-selectin-dependent rolling/tethering and transition to LFA-1-mediated arrest were not affected. Mst1^−/− lymphocytes were also defective in the stabilization of adhesion through α4 integrins. Consequently, Mst1^−/− mice had hypotrophic peripheral lymphoid tissues and reduced marginal zone B cells and dendritic cells in the spleen, and defective emigration of single positive thymocytes. Furthermore, Mst1^−/− lymphocytes had impaired motility over lymph node-derived stromal cells and within lymph nodes. Thus, our data indicate that Mst1 is a key enzyme involved in lymphocyte entry and interstitial migration.     

Vasoactive intestinal polypeptide stimulates cyclic AMP production in mouse N1E-115 neuroblastoma cells: Modulation by a protein kinase C activator and ionomycin

In this study, we investigated the vasoactive intestinal polypeptide (VIP)-stimulated cAMP production and its interaction with protein kinase C activation and elevation of intracellular Ca²⁺ in N1E-115 neuroblastoma cells. VIP treatment caused a 55-fold increase in cAMP accumulation. Addition of 4β-phorbol 12-myristate 13-acetate reduced VIP-but not forskolin-stimulated cAMP response. In comparison, ionomycin potentiated both VIP- and forskolin-induced cAMP accumulation. Our results indicate that VIP stimulates cAMP accumulation in N1E-115 cells, and that although activation of protein kinase C inhibits the VIP-stimulated cAMP response, elevation of intracellular Ca²⁺ potentiates this signaling pathway.     

Comparative study of intracellular glutathione content in rat lymphocyte cultures treated with 2-mercaptoethanol and interleukin-2

The level of intracellular glutathione (GSH) in mitogen-stimulated mouse lymphocytes is increased in the presence of 2-mercaptoethanol (2-ME), an enhancer of lymphocyte activation and proliferation. Since proliferation of lymphocytes in response to mitogens involves direct activation by a mitogen followed by continued proliferation in response to interleukin-2 (IL-2), we have investigated the effect of 2-ME and exogenous IL-2 on the GSH content and cell proliferation of rat lymphocytes stimulated with phytohemagglutinin (PHA). PHA stimulation increased both GSH content and the magnitude of the proliferative response, as measured by thymidine incorporation into cellular DNA. However, incubation of stimulated lymphocytes with 2-ME or IL-2 for 72 hr produced a significant further elevation of GSH levels and thymidine incorporation. 2-ME also increased the GSH content in unstimulated cultures, but it had little effect on thymidine incorporation. IL-2 increased GSH content and decreased thymidine incorporation in unstimulated lymphocytes. Exposure of cells to DL-buthionine-(S,R)-sulfoximine (BSO), an inhibitor of GSH biosynthesis, significantly depleted GSH and lowered the proliferative response, suggesting a crucial role of de novo GSH synthesis for lymphocyte activation. The data suggest that both 2-ME and IL-2 promote lymphocyte proliferation, although the mechanisms by which intracellular GSH levels are increased by the agents are apparently different.Copies of articles are available through ISI Document Delivery Services c/o The Genuine Article, 3501 Market Street, Philadelphia, PA 19104.     

Regulation of gene expression by the intracellular second messengers IP3 and diacylglycerol

In Dictyostelium, extracellular cAMP interacts specifically with cell-surface receptors to promote the accumulation of a variety of intracellular second messengers, such as 3'-5' cyclic adenosine monophosphate (cAMP) and 1,4,5 inositol trisphosphate (IP3). We and others have shown that activation of the cell-surface cAMP receptor can also modulate the expression of the Dictyostelium genome during development. In at least one instance, synthesis of intracellular cAMP is required for appropriate gene regulation. However, the induction of most cAMP-dependent gene expression can occur in the absence of receptor-mediated activation of adenylate cyclase and a consequent accumulation of intracellular cAMP. These results suggest that other intracellular second messengers produced in response to receptor activation may potentially act as signal transducers to modulate gene expression during development. In vertebrate cells, IP3 and diacylglycerol (DAG) are intracellular activators of specific protein kinases; they are produced in equimolar amounts by cleavage of phosphoinositol bisphosphate after a receptor-mediated activation of a membrane-bound phosphodiesterase. IP3 and, thus, by inference, diacyl-glycerol are synthesized in Dictyostelium as a response to cAMP interacting with its cell-surface receptor. Using defined conditions to inhibit the accumulation of extracellular cAMP, we have examined the effects of these compounds on the expression of genes that require cAMP for their maximal expression. Our results suggest that intracellular IP3 and DAG may in part mediate the action of extracellular cAMP on the expression of the Dictyostelium genome.     

Microtubule Depolymerization Induces Stress Fibers,Focal Adhesions,and DNA Synthesis via the GTP-Binding Protein Rho

Microtubule depolymerization has multiple consequences that include actin stress fiber and focal adhesion assembly, increased tyrosine phosphorylation and DNA synthesis. Similar effects induced by serum, or agents such as lysophosphatidic acid, have previously been shown to be mediated by the GTP-binding protein Rho. We have investigated whether the effects of microtubule depolymerization are similarly mediated by Rho and show that they are blocked by the specific Rho inhibitor, C3 transferase. Because microtubule depolymerization induces these effects in quiescent cells, in which Rho is largely inactive, we conclude that microtubule depolymerization leads to activation of Rho. The activation of Rho in response to microtubule depolymerization and the consequent stimulation of contractility suggest a mechanism by which microtubules may regulate microfilament function in various motile phenomena. These range from growth cone extension to the development of the contractile ring during cytokinesis, in which there are interactions between the microtubule and microfilament systems.     

Interplay between exchange protein directly activated by cAMP (Epac) and microtubule cytoskeleton

"Exchange protein directly activated by cAMP" (Epac) is a newly discovered cAMP receptor that mediates the intracellular cAMP actions in addition to the classic cAMP-dependent protein kinase system. In this study, we show that Epac interacts directly with tubulin, co-purifies with cellular microtubules, and co-localizes with the mitotic spindle assembly. Association with microtubules suppresses Epac-mediated Rap1 activation, while the binding of Epac promotes microtubule formation. These results demonstrate that Epac plays an important role in connecting the microtubule cytoskeleton network and intracellular cAMP-signalling.     

Effect of adrenaline on lymphocyte metabolism and function. A mechanism involving cAMP and hydrogen peroxide

This study examined the effect of adrenaline on lymphocyte metabolism and function. The following parameters were addressed: cell proliferation, glucose and glutamine metabolism as indicated by the measurement of enzyme activities, the utilization of metabolites and production and oxidation of substrates. We also evaluated the involvement of beta-receptors in this response as well as the possible effect of cAMP and hydrogen peroxide in the process of lymphocyte activation by adrenaline. The results indicated that adrenaline is able to induce metabolic changes in lymphocytes that are related to enhanced proliferative capacity, but under physiological conditions fails to initiate the process, the catecholamine could, increase cell proliferation via increased production of H₂O₂ by macrophages, since this reactive oxygen intermediate can act as a trigger for lymphocyte activation. The results also showed that distinct populations of lymphocytes present different responses to adrenaline activation, as demonstrated by cells obtained from the same site but exposed to different mitogens such as LPS and ConA. © 1997 John Wiley & Sons, Ltd.     

Interactions between parathyroid hormone and prostaglandins on renal cortical cyclic AMP

Effects of parathyroid hormone (PTH) and several prostaglandins (PGs) on cyclic AMP (cAMP) metabolism were studied and compared in isolated renal cortical tubules from male hamsters. Both production and intracellular degradation of cAMP were increased by PTH and each of the PGs tested (PGE₂, PGE₁, PGI₂). Production of cAMP was increased to similar levels by maximal concentrations of PTH and each PG, however, degradation of cAMP was significantly higher in response to PTH than with any of the PGs. This difference in intracellular degradation of cAMP was responsible for the much higher concentrations of cAMP in renal cortical tubules exposed to PGs (PGE₁, PGE₂, PGI₂) than to PTH. Submaximal amounts of each PG produced additive increases in cAMP concentrations in the presence of maximal amounts of PTH. Additivity of the combined responses was lost, however, as the PGs concentrations reached their maximas. The results suggest that renal PGs (PGE₂ and PGI₂) may modulate the effects of PTH on cAMP concentrations in renal cortical tubules.     

Biochemical basis of HLA-DR and CR3 modulation on human peripheral blood monocytes by lipopolysaccharide

The biochemical events leading to enhanced membrane expression of HLA-DR and CR3 by human peripheral blood monocytes (MO) following exposure to bacterial lipopolysaccharide (LPS) were examined. In a previous study we demonstrated that an increase in intracellular calcium was necessary, but not sufficient, for MO to increase membrane expression of both antigens within 1 hr of addition of LPS. The present study was initiated to examine the other biochemical requirements which lead to the MO response to LPS. Enhanced expression of both antigens following addition of LPS was dependent on microfilament function, but independent of microtubule function and of protein synthesis. Inhibition of formation of cyclooxygenase or lipoxygenase metabolites of arachidonic acid had no effect on HLA-DR or CR3 modulation by LPS. A role for phosphatidylinositol metabolism was suggested by the inhibition of the MO response to LPS by dibutyryl cAMP and theophylline and by the enhanced expression of both antigens following addition of phorbol diesters. However, H-7, a putative inhibitor of protein kinase C, did not alter the MO response to LPS or phorbol diesters. These results suggest that LPS enhances expression of HLA-DR and CR3 by inducing redistribution of these antigens from an intracellular pool. The data also support a role for the generation of hydrolysis products of phosphatidylinositol, leading to calcium redistribution and activation of protein kinase C or other kinases, in the MO response to LPS.     

Agents that raise cAMP in human T lymphocytes release an intracellular pool of calcium in the absence of inositol phosphate production

Prostaglandins (PGs) of the E series are recognized by specific receptors on T lymphocytes which lead to an increase in cAMP. The role of cAMP in modulation of T lymphocyte function is unknown. Here, we demonstrate that agents which increase cAMP in human T cells raise the intracellular free calcium concentration ([Ca2+]i). This increase in [Ca2+]i occurred following receptor stimulation with PGEs or by bypassing the receptor with the cell-permeant analog 8-(4-chlorophenylthio)-cAMP or forskolin, a direct activator of adenylyl cyclase. The calcium response to a submaximally stimulatory concentration of PGE2 was potentiated by the cAMP phosphodiesterase inhibitor isobutylmethylxanthine. A time course of cAMP production in response to PGE2 stimulation closely resembled the calcium response and suggested that the two events were coincident. The PGE2 concentrations required to achieve 50% maximum effect of cAMP production and increases in [Ca2+]i were similar, 0.07 and 0.15 microM respectively. Chelation of extracellular Ca2+ did not abolish the PGE2-stimulated Ca2+ response, suggesting that an intracellular source of calcium was sensitive to cAMP. Significant inositol phosphate production was not detected in response to PGE2 over a wide concentration range. The PGE2-induced calcium response curves were of lesser magnitude with shorter times to peak than those of a known inositol 1,4,5 trisphosphate-producing agonist, anti-CD3, suggesting distinct Ca2+ release mechanisms. However, the cAMP-releasable store appeared to be contained within the inositol trisphosphate-releasable store since no response could be seen with cAMP-elevating agents following emptying of the inositol trisphosphate-sensitive pool of Ca2+.