Free cAMP Research Articles

Regulated expression of HCN channels and cAMP levels shape the properties of the h current in developing rat hippocampus

The hyperpolarization-activated current (I(h)) contributes to intrinsic properties and network responses of neurons. Its biophysical properties depend on the expression profiles of the underlying hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels and the presence of cyclic AMP (cAMP) that potently and differentially modulates I(h) conducted by HCN1, HCN2 and/or HCN4. Here, we studied the properties of I(h) in hippocampal CA1 pyramidal cells, the developmental evolution of the HCN-subunit isoforms that contribute to this current, and their interplay with age-dependent free cAMP concentrations, using electrophysiological, molecular and biochemical methods. I(h) amplitude increased progressively during the first four postnatal weeks, consistent with the observed overall increased expression of HCN channels. Activation kinetics of the current accelerated during this period, consonant with the quantitative reduction of mRNA and protein expression of the slow-kinetics HCN4 isoform and increased levels of HCN1. The sensitivity of I(h) to cAMP, and the contribution of the slow component to the overall I(h), decreased with age. These are likely a result of the developmentally regulated transition of the complement of HCN channel isoforms from cAMP sensitive to relatively cAMP insensitive. Thus, although hippocampal cAMP concentrations increased over twofold during the developmental period studied, the coordinated changes in expression of three HCN channel isoforms resulted in reduced effects of this signalling molecule on neuronal h currents.

Dual Roles of P2 Purinergic Receptors in Insulin-stimulated Leptin Production and Lipolysis in Differentiated Rat White Adipocytes

ATP is co-localized with norepinephrine at the sympathetic nerve terminals and may be released simultaneously upon neuronal stimulation, which results in activation of purinergic receptors. To examine whether leptin synthesis and lipolysis are influenced by P2 purinergic receptor activation, the effects of ATP and other nucleotides on leptin secretion and glycerol release have been investigated in differentiated rat white adipocytes. Firstly, insulin-induced leptin secretion was inhibited by nucleotide treatment with the following efficacy order: 3'-O-(4-benzoyl)benzoyl ATP (BzATP) > ATP >> UTP. Secondly, treatment of adipocytes with ATP increased both intracellular Ca(2+) concentration and cAMP content. Intracellular calcium concentration was increased by ATP and UTP, but not BzATP, an effect attributed to phospholipase C-coupled P2Y(2). On the other hand, cAMP was generated by treatment with BzATP and ATPgammaS, but not UTP, indicating functional expression of adenylyl cyclase-coupled P2Y(11) receptors in white adipocytes. Thirdly, lipolysis was significantly activated by BzATP and ATP, which correlated with the characteristics of the P2Y(11) subtype. Taken together, the data presented here suggest that white adipocytes express at least two different types of P2Y receptors and that activation of P2Y(11) receptor might be involved in inhibition of leptin production and stimulation of lipolysis, suggesting that purinergic transmission can play an important role in white adipocyte physiology.

Novel Single Chain cAMP Sensors for Receptor-induced Signal Propagation

cAMP is a universal second messenger of many G-protein-coupled receptors and regulates a wide variety of cellular events. cAMP exerts its effects via cAMP-dependent protein kinase (PKA), cAMP-gated ion channels, and two isoforms of exchange protein directly activated by cAMP (Epac). Here we report the development of novel fluorescent indicators for cAMP based on the cAMP-binding domains of Epac and PKA. Fluorescence resonance energy transfer between variants of green fluorescent protein (enhanced cyan fluorescent protein and enhanced yellow fluorescent protein) fused directly to the cAMP-binding domains was used to analyze spatial and temporal aspects of cAMP-signaling in different cells. In contrast to previously developed PKA-based indicators, these probes are comprised of only a single binding site lacking cooperativity, catalytic properties, and interactions with other proteins and thereby allow us to easily image free intracellular cAMP and rapid signaling events. Rapid beta-adrenergic receptor-induced cAMP signals were observed to travel with high speed ( approximately 40 microm/s) throughout the entire cell body of hippocampal neurons and peritoneal macrophages. The developed indicators could be ubiquitously applied to studying cAMP, its physiological role and spatio-temporal regulation.

Coordinate regulation of membrane cAMP by Ca2+-inhibited adenylyl cyclase and phosphodiesterase activities.

Activation of store-operated Ca(2+) entry inhibits type 6 adenylyl cyclase (EC; AC(6); Yoshimura M and Cooper DM. Proc Natl Acad Sci USA 89: 6712-6720, 1992) activity in pulmonary artery endothelial cells. However, in lung microvascular endothelial cells (PMVEC), which express AC(6) and turn over cAMP at a rapid rate, inhibition of global (whole cell) cAMP is not resolved after direct activation of store-operated Ca(2+) entry using thapsigargin. Present studies sought to determine whether the high constitutive phosphodiesterase activity in PMVECs rapidly hydrolyzes cAMP so that Ca(2+) inhibition of AC(6) is difficult to resolve. Direct stimulation of adenylyl cyclase using forskolin and inhibition of type 4 phosphodiesterases using rolipram increased cAMP and revealed Ca(2+) inhibition of AC(6). Enzyme activity was assessed using PMVEC membranes, where Ca(2+) and cAMP concentrations were independently controlled. Endogenous AC(6) activity exhibited high- and low-affinity Ca(2+) inhibition, similar to that observed in C6-2B cells, which predominantly express AC(6). Ca(2+) inhibition of AC(6) in PMVEC membranes was observed after enzyme activation and inhibition of phosphodiesterase activity and was independent of the free cAMP concentration. Thus, under basal conditions, the constitutive type 4 phosphodiesterase activity rapidly hydrolyzes cAMP so that Ca(2+) inhibition of AC(6) is difficult to resolve, indicating that high phosphodiesterase activity works coordinately with AC(6) to regulate membrane-delimited cAMP concentrations, which is important for control of cell-cell apposition.

Type II beta regulatory subunit of cAMP-dependent protein kinase: purification strategies to optimize crystallization.

To elucidate the structural basis for important differences between types I and II regulatory subunit isoforms (RI and RII) of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase, the full-length RII beta isoform and five RII beta deletion mutants were constructed, expressed, purified, and screened for crystallization. Only one of these six proteins yielded diffraction quality crystals. Crystals were grown of the RII beta deletion mutant (delta 1-111) monomer potentially in complex with two cAMP molecules. X-ray diffraction quality data were obtained only after significant modification to existing purification procedures. Modifications required a Sepharose, not agarose, support for cAMP affinity chromatography followed by rapid, quantitative removal of free cAMP by size-exclusion chromatography under reducing conditions. Data to 2.4 A resolution were collected at 29 degrees C using synchrotron radiation on a single crystal measuring 0.2 x 0.3 x 1.2 mm(3). Data were 99% complete. The hexagonal crystal belonged to space group P6((1)) or P6((5)) with unit cell dimensions a = b = 161.62 A and c = 39.66 A.

Increased plasma free cyclic-AMP levels following major trauma and their relevance to the immune response

Background: Following injury, neutrophil mobilisation is an important element of the immune response. The ideal features of an agent responsible for this mobilisation would include the ability to mobilise neutrophils without activating them, and rapid reversibility. This study investigates the hypothesis that raised levels of plasma adrenaline following trauma act via cyclic adenosine monophosphate (cAMP) to mobilise neutrophils, and measures the amount of cAMP extruded from cells into the plasma following injury. Methods: 20-ml samples of venous blood were drawn from 34 trauma patients within 3 h of injury and divided between three sample tubes: (1) ethylene diamine tetra-acetic acid anticoagulant (EDTA) for full blood count; (2) cooled EDTA for cAMP levels; and (3) cooled lithium heparin for catecholamines. The latter two tubes were immediately centrifuged at low temperature and the supernatant plasma deep frozen pending analysis. Adrenaline was measured using high pressure liquid chromatography (HPLC) and cAMP measured by an enzyme immunoassay technique. Results: 34 patients, six of whom had sustained minor trauma (ISS 1–8), 12 moderate trauma (ISS 9–15) and 16, major trauma (ISS 16 and above) were studied. Median age was 39 years (range 16–77) and 30 patients were male. Plasma adrenaline levels were available for 28 of the patients. Plasma free cAMP levels were significantly raised in patients with major trauma ( P<0.006). There were positive correlations between the plasma levels of cAMP and adrenaline (rho 0.660, P=0.011), adrenaline and neutrophil count (rho 0.654, P=0.01) and cAMP and neutrophil count (rho 0.508, P=0.013). Conclusions: Evidence is presented of the possible inter-relationships between neutrophil counts, adrenaline levels and cAMP levels following injury, supporting the proposition that neutrophil mobilisation pathways are activated early via β-adrenergic stimulation.

Differential effect of thyroid-stimulating hormone (TSH) on intracellular free calcium and cAMP in cells transfected with the human TSH receptor.

The thyroid-stimulating hormone (TSH) binds to a receptor which activates adenylate cyclase and elevates cAMP concentration. In addition, effects of TSH on intracellular calcium and inositol phosphate accumulation have been reported. However, the mechanism of TSH-stimulated accumulation of inositol phosphates and elevation of calcium levels is unresolved. Previous work from this laboratory has shown TSH to cause acute transient increases in intracellular calcium in pig, human and FR TL-5 rat thyroid cells as well as in cell transfected with the human TSH receptor (JPO9 cells) in some (but not all) experiments. The aim of this study was to investigate the variability of the calcium response to TSH in JPO9 cells to learn more about the nature of this calcium signal induction. Calcium responses to TSH were determined using the fluorochrome fura-2 in both monolayers of adherent cells and adherent single cells. The responses to a single addition and to repetitive additions of TSH were compared. We also determined the cAMP response to TSH using these two protocols of TSH addition. Our data show that, whereas the cAMP response to TSH is highly predictable and consistent and does not require multiple exposures to TSH, cells were unlikely to respond to TSH with an increase in calcium unless they received multiple challenges with the hormone. A single addition of 10 mU/ml TSH failed to increase calcium in any of 40 single cells examined and in only 4 of 15 monolayers of cells (27%) examined; in contrast, 10 of 12 monolayers eventually responded with an increase in calcium after multiple exposure to TSH and 18 of 67 single cells. Similar data were obtained whether calcium was measured in single cells or in populations of cells. We also demonstrated cooperativity between an adenosine derivative, N6-(L-2-phenylisopropyl)adenosine, and TSH such that their co-administration resulted in a consistent and marked elevation in calcium levels not achieved with either agonist alone. In summary, we suggest that the coupling between the TSH receptor and the intracellular signalling system that leads to activation of intracellular calcium in JPO9 cells requires repetitive stimulation or the influence of other agonists, in contrast with the coupling between the TSH receptor and activation of the adenylate cyclase enzyme.

Inhibition of inositol 1,4,5-trisphosphate-induced Ca2+ release by cAMP-dependent protein kinase in a living cell.

Interaction of intracellular free calcium ([Ca2+]i) and cAMP signaling mechanisms was examined in intact single megakaryocytes by using a combination of single-cell fluorescence microscopy to measure [Ca2+]i and flash photolysis of caged Ca2+, inositol 1,4, 5-trisphosphate (IP3), or cAMP to elevate rapidly the concentration of these compounds inside the cell. Photolysis of caged IP3 stimulated Ca2+ release from an IP3-sensitive store. The cAMP-elevating agent carbacyclin inhibited this IP3-induced rise in [Ca2+]i but did not affect the rate of Ca2+ removal from the cytoplasm after photolysis of caged Ca2+. Photolysis of caged cAMP during ADP-induced [Ca2+]i oscillations caused the [Ca2+]i oscillation to transiently cease without affecting the rate of Ca2+ uptake and/or extrusion. We conclude that the principal mechanism of cAMP-dependent inhibition of Ca2+ mobilization in megakaryocytes appears to be by inhibition of IP3-induced Ca2+ release and not by stimulation of Ca2+ removal from the cytoplasm. Two inhibitors of cAMP-dependent protein kinase, a specific peptide inhibitor of the catalytic subunit of cAMP protein kinase and KT5720, blocked the inhibitory effect of carbacyclin, indicating that the inhibition of IP3-induced Ca2+-release by carbacyclin is mediated by cAMP-dependent protein kinase.

DOPAMINE AS A MODULATOR OF IONIC TRANSPORT AND NA+/K+-ATPASE ACTIVITY IN THE GILLS OF THE CHINESE CRAB ERIOCHEIR SINENSIS

ABSTRACT Dopamine and dibutyryl cAMP induced a significant increase of 22Na+ influx in salt-transporting posterior gills isolated from the Chinese crab Eriocheir sinensis acclimated to fresh water. When isolated gills were incubated and perfused in Cl--free saline (Cl replaced by gluconate), dopamine or dibutyryl cAMP still produced a significant increase of 22Na+ influx. After 1-h perfusion, dopamine increased intracellular cAMP content and Na+/K+-ATPase activity as compared with control perfusions without dopamine.

Free cyclic AMP increases in PC12 cells on depolarization.

The temporal dynamics of the intracellular second messenger cyclic AMP (cAMP) were monitored in living PC12 cells by digital fluorescence ratio imaging using FlCRh, a single-excitation dual-emission cAMP indicator. When the cells were depolarized by exposure to high K+, the free cAMP concentration was elevated, and then slowly decreased back to resting levels when the depolarizing stimulus was removed. Furthermore, the cAMP elevation due to depolarization decreased with successive depolarizations.

Dual signal transduction through delta opioid receptors in a transfected human T-cell line.

Opiates are known to function as immunomodulators, in part by effects on T cells. However, the signal transduction pathways mediating the effects of opiates on T cells are largely undefined. To determine whether pathways that regulate free intracellular calcium ([Ca2+]i) and/or cAMP are affected by opiates acting through delta-type opioid receptors (DORs), a cDNA encoding the neuronal DOR was expressed in a stably transfected Jurkat T-cell line. The DOR agonists, deltorphin and [D-Ala2, D-Leu5]-enkephalin (DADLE), elevated [Ca2+]i, measured by flow cytofluorometry using the calcium-sensitive dye, Fluo-3. At concentrations from 10(-11)-10(-7) M, both agonists increased [Ca2+]i from 60 nM to peak concentrations of 400 nM in a dose-dependent manner within 30 sec (ED50 of approximately 5 x 10(-9) M). Naltrindole, a selective DOR antagonist, abolished the increase in [Ca2+]i, and pretreatment with pertussis toxin was also effective. To assess the role of extracellular calcium, cells were pretreated with EGTA, which reduced the initial deltorphin-induced elevation of [Ca2+]i by more than 50% and eliminated the second phase of calcium mobilization. Additionally, the effect of DADLE on forskolin-stimulated cAMP production was determined. DADLE reduced cAMP production by 70% (IC50 of approximately equal to 10(-11) M), and pertussis toxin inhibited the action of DADLE. Thus, the DOR expressed by a transfected Jurkat T-cell line is positively coupled to pathways leading to calcium mobilization and negatively coupled to adenylate cyclase. These studies identify two pertussis toxin-sensitive, G protein-mediated signaling pathways through which DOR agonists regulate the levels of intracellular messengers that modulate T-cell activation.

Modulation of free intracellular calcium and cAMP by morphine and cannabinoids, alone and in combination in mouse brain and spinal cord synaptosomes

Changes in [Ca ++] i and cAMP were evaluated as possible mechanisms by which the cannabinoids enhance the antinociception of morphine. The addition of subactive concentrations of Δ9- (THC) and morphine in combination to brain synaptosomes did not result in an enhanced decrease in [Ca ++] i; however, this drug combination enhanced decreases in [Ca ++] i in spinal cord synaptosomes. The combination of CP55,940 and morphine produced enhanced decreases in [Ca ++] i in both brain and spinal cord synaptosomes. In brain synaptosomes, the combination of Δ9-THC and morphine produced an additive decrease in cAMP accumulation, whereas no significant change was observed with this combination in the spinal cord. Thus, the difference in the modulation of [Ca ++] i but not cAMP in the brain in vitro may be a predictor of the greater-than-additive antinociceptive effects observe in vivo.

Stress relaxation of fibroblasts activates a cyclic AMP signaling pathway.

Mechanical force regulates gene expression and cell proliferation in a variety of cell types, but the mechanotransducers and signaling mechanisms involved are highly speculative. We studied the fibroblast signaling mechanism that is activated when cells are switched from mechanically stressed to mechanically relaxed conditions, i.e., stress relaxation. Within 10 min after initiation of stress relaxation, we observed a transient 10-20-fold increase in cytoplasmic cyclic AMP (cAMP) and a threefold increase in protein kinase A activity. The increase in cAMP depended on stimulation of adenylyl cyclase rather than inhibition of phosphodiesterase. Generation of cAMP was inhibited by indomethacin, and release of arachidonic acid was found to be an upstream step of the pathway. Activation of signaling also depended on influx of extracellular Ca2+ because addition of EGTA to the incubations at concentrations just sufficient to exceed Ca2+ in the medium inhibited the stress relaxation-dependent increase in free arachidonic acid and cAMP. This inhibition was overcome by adding CaCl2 to the medium. On the other hand, treating fibroblasts in mechanically stressed cultures with the calcium ionophore A23187-stimulated arachidonic acid and cAMP production even without stress relaxation. In summary, our results show that fibroblast stress relaxation results in activation of a Ca(2+)-dependent, adenylyl cyclase signaling pathway. Overall, the effect of stress relaxation on cAMP and PKA levels was equivalent to that observed after treatment of cells with forskolin.

TSH action on cAMP binding to the regulatory subunits of cAMP-dependent protein kinases in pig thyroid cell cultures

This study examines the mechanism of TSH action on the cAMP-dependent protein kinases (PKA) by measuring the catalytic activity of the two PKA isozymes (PKA I and PKA II) and their capacity to bind cAMP to the regulatory subunits (RI and RII) in thyroid cell cultures exposed for two days to different doses of TSH. In TSH-treated cell cultures a selective down regulation (up to 60%) of the catalytic activity was found; the PKA I was down regulated at lower TSH doses (0.1 mU/ml and even 0.05 mU/ml) than was the PKA II (1.0 mU/ml TSH). At the dose of 1.0 mU/ml the loss of the catalytic activity in PKA I and PKA II was respectively 60% and 40%. No free catalytic activity was found either in control or in TSH-treated cells. Binding of cAMP to regulatory subunits (R) measured under exchange conditions at 37°C, showed that no change in total regulatory subunit protein content occurs in TSH-treated cells. Binding of cAMP to R subunits at 4°C (when only free cAMP binding sites are measured) revealed an important endogenous occupancy of cAMP binding sites of RI and RII isoreceptors under basal conditions (40%) and a significantly increased occupancy after exposure of cells to TSH (60%). Pools of regulatory subunits with more than 50% of sites occupied, which were devoid of enzyme activity, were found both, in control and TSH-exposed cells. They were identified as RI subunits which represented a mixed population of native and partly degraded molecules. In summary, in thyroid cell cultures, TSH when chronically present, has an independent action on the catalytic and the regulatory subunits of PKAs. The catalytic subunit activity was down regulated in parallel with an increased residual cAMP binding to regulatory subunits, but with no change in total regulatory subunit protein. The increased fractional saturation of cAMP-binding sites in TSH-treated cells renders their PKAs more sensitive to further stimulations and may help understand the molecular mechanism of the tonic effect of TSH on thyroid cells in culture.

Spreading of HeLa cells on a collagen substratum requires a second messenger formed by the lipoxygenase metabolism of arachidonic acid released by collagen receptor clustering.

HeLa cells attach to a variety of substrata but spread only on collagen or gelatin. Spreading is dependent on collagen-receptor upregulation, clustering, and binding to the cytoskeleton. This study examines whether second messengers are involved in initiating the spreading process on gelatin. The levels of cytosolic free calcium ([Ca++]i), cAMP, and cytoplasmic pH (pHi) do not change during cell attachment and spreading. However, a basal level of [Ca++]i and an alkaline pH(i) are required for spreading. There is an activation of protein kinase C (PKC) and a release of arachidonic acid (AA) on attachment and before cell spreading. Inhibition of PKC does not block cell spreading, indicating that PKC activation is not essential for spreading. Inhibition of phospholipase A2 blocks cell spreading, whereas addition of exogeneous AA overcomes this inhibitory effect. Among AA metabolic pathways, inhibitors of lipoxygenase (LOX) block cell spreading, suggesting that a LOX product(s) formed from AA initiates spreading. Clustering receptors for collagen with polyclonal antibodies, or with anti-collagen-receptor antigen-binding fragments (Fab) in combination with a secondary antibody, induce AA release. Also, AA is released when cells attach to either immobilized gelatin or immobilized Arg-Gly-Asp (RGD) peptide. Thus, AA is released whenever receptor clustering is observed. Receptor occupancy is not sufficient to release AA; when cells are treated with gelatin or RGD peptide in solution or anti-collagen-receptor Fab fragments without secondary antibody, conditions where receptor clustering is not observed, AA is not released. Thus, a LOX metabolite(s) of AA formed by collagen-receptor clustering is a second messenger(s) that initiates HeLa cell spreading. LOX inhibitors also block the spreading of bovine aortic endothelial cells, chicken embryo fibroblasts, and CV-1 fibroblasts on gelatin or fibronectin, indicating that other cells might use the same second messenger system in initiating cell-substratum adhesion.

Effect of testosterone on cancellous bone density during growth in the rabbit

The effects of three synthetic amino-terminal fragments of human parathyroid hormone-related peptide (hPTHrP) on cytosolic free calcium ([Ca2+]i) mobilization and intracellular cAMP production were investigated and compared with those of human PTH(1-34) in opossum kidney (OK) cells. The mean basal [Ca2+]i in OK cells was 115 ± 7.8 nM (mean ± SE, n = 30). Human PTHrP(1-34), [40Tyr]hPTHrP(1-40) and hPTH(1-34) significantly (P < 0.01) raised [Ca2+]i to 149 ± 3, 150 ± 5 and 148 ± 6% of the basal value, respectively (mean ± SE, n = 3). These three peptides showed similar dose dependency at concentrations between 10−10 and 10−7 M. These increases appeared to be, in part, due to mobilization from intracellular calcium stores. On the other hand, these three peptides also stimulated intracellular cAMP production in an equipotent manner in OK cells. However, both 10−3 M dibutyryl cAMP and 10−5 M forskolin failed to cause an increase in [Ca2+]i. Furthermore, [40Tyr]hPTHrP(3-40) neither affected the intracellular cAMP content significantly nor caused an increase in [Ca2+]i. These results indicate that hPTHrP mimics PTH by activating a dual second messenger system ([Ca2+]i and cAMP) in OK cells, and that [Ca2+]i stimulation by hPTHrP and hPTH is independent of cAMP production. Additionally, N-terminal amino acid position 1 to 2 of hPTHrP appears to play a crucial role in both [Ca2+]i stimulation and cAMP production.

Influence of endothelin on human platelet aggregation and prostacyclin generation from human vascular endothelial cells in culture.

The effects of endothelin (ET) on the function of cultured human umbilical vein endothelial cells (HUVEC) and that of human platelets were investigated with reference to endothelium-derived relaxing factor (EDRF) and PGI2. Considering the platelets, ET had no effect on platelet-rich plasma (PRP) aggregation, the generation of thromboxane A2 ([TXA2]) from platelets, and cytosolic free calcium ion concentration ([Ca++]i), cAMP content ([cAMP]i) or cGMP content ([cGMP]i) in platelets. In contrast, the addition of the solution in which HUVEC had been incubated with ET to PRP produced a decrease in PRP aggregation, [TXA2], and [Ca++]i, and an increase not only in [cAMP]i but also in [cGMP]i in platelets. In the HUVEC pretreated with acetylsalicylic acid (aspirin), this increase of [cGMP]i was not affected, but the HUVEC-mediated decrease in PRP aggregation, [TXA2], and [Ca++]i induced by ET were not completely abolished. However, the pretreatment of HUVEC with a combination of aspirin and L-NG-monomethyl arginine (LNMMA) as an inhibitor of EDRF completely abolished the HUVEC-mediated decrease in PRP aggregation, [TXA2] and [Ca++]i induced by ET, and also abolished the enhancement of [cGMP]i and [cAMP]i in platelets. The PGI2 of HUVEC was enhanced by ET with no changes in [Ca++]i, [cAMP]i and [cGMP]i. The ET-induced enhancement was remarkably attenuated by pretreating the HUVEC with aspirin, but not with LNMMA. We conclude that ET attenuates the aggregation of platelets through a decrease in [TXA2] by an increase in [cAMP]i via the increase in PGI2 of HUVEC, and by an increase in [cGMP]i via EDRF.

Mechanisms of cAMP-mediated relaxation of distal circular muscle in rabbit colon.

Photolytic release of free adenosine 3',5'-cyclic monophosphate (cAMP) from its caged form was used to evaluate the physiological role of several proposed mechanisms of cAMP-mediated relaxation of circular smooth muscle in the distal rabbit colon. Photolysis of caged cAMP produced a rapid relaxation of bethanechol-contracted distal circular muscle strips that was dependent on ultraviolet exposure time. An increase in release of free cAMP, associated with increased ultraviolet exposure, was confirmed with high-performance liquid chromatography. Vanadate (an ATPase inhibitor) (3 mM) caused a 48% decrease in cAMP-mediated relaxation, while ouabain and a zero K+ bath solution failed to affect relaxation. cAMP-mediated relaxation of KCl-contracted strips was significantly less effective than that of bethanechol-contracted strips. Although this finding suggested that cAMP-mediated relaxation may involve K+ channel modulation, specific (glibenclamide, charybdotoxin) and nonspecific (TEA) K+ channel blockade failed to affect cAMP-mediated relaxation of bethanechol-contracted strips. The photolytic release of cAMP failed to relax Ca(2+)-contracted saponin skinned muscle strips. These studies suggest 1) modulation of Ca2+ pumps plays an important role in this model of relaxation of distal colonic circular muscle in the rabbit colon, 2) modulation of the Na+ pump or sarcolemmal K+ channels may not play an important physiological role in relaxation induced by a rapid rise in intracellular cAMP, and 3) cAMP does not seem to have a significant physiological effect on the Ca2+ sensitivity contractile apparatus.

Growth of the marine luminous bacterium Vibrio fischeri on 3':5'-cyclic AMP: correlation with a periplasmic 3':5'-cyclic AMP phosphodiesterase

Summary: The marine luminous bacterium Vibrio fischeri, the species-specific light-organ symbiont of monocentrid (pinecone) fish, grew on 3′:5′-cyclic adenosine monophosphate (cAMP) as a sole source of carbon and energy, a capability not previously described in bacteria. In a minimal, chemically defined medium containing cAMP as the sole source of carbon and energy, V. fischeri cells grew more slowly than on glucose or ribose, but as quickly as on 5′-AMP. Expression of luminescence, which is dependent on cAMP in V. fischeri, was stimulated in cells grown on cAMP compared to cells grown on glucose or ribose. All strains of V. fischeri tested (MJ-1, B-61, ATCC 7744, MJ-A1, CG-A1) grew on cAMP, as did strains of two other marine luminous bacteria, V. logei and Photobacterium phosphoreum, and strains of the terrestrial enteric bacterium Serratia marcescens. Other tested species of marine luminous bacteria (P. leiognathi, Shewanella [Alteromonas] hanedai, V. harveyi, V. orientalis, V. splendidus) and terrestrial enteric bacteria (Escherichia coli, Salmonella typhimurium), which grew on 5′-AMP or ribose, did not grow on cAMP. Assays on intact cells and periplasmic extracts revealed that V. fischeri MJ-1 produced a periplasmic 3′:5′-cAMP phosphodiesterase (cAMP phosphodiesterase) of very high specific activity [9·0 μmol phosphate released (mg protein)−1 min−1] and narrow substrate specificity (3′:5′-cAMP and 3′:5′-cGMP were attacked). The novel periplasmic location and unusually high activity of cAMP phosphodiesterase appear to account for the ability of this species to grow on cAMP. The periplasmic cAMP phosphodiesterase of V. fischeri night play a role in degrading free cAMP in seawater or in a cAMP-mediated aspect of the light organ symbiosis with monocentrid fish.

Prostaglandins enhance parathyroid hormone-evoked increase in free cytosolic calcium concentration in osteoblast-like cells

Prostaglandins (PGs) are autocrine or paracrine hormones that may interact with circulating hormones such as parathyroid hormone (PTH) in bone. We examined the interaction of the PGs, PGF 2α, PGE 2, and 6-keto-PGF 1α with PTH to enhance the rapid, initial transient rise in free cytosolic calcium ([Ca 2+] i) and cAMP levels stimulated by PTH. Pretreatment of UMR-106, MC3T3-E1, and neonatal rat calvarial osteoblast-like cells by PGs resulted in an enhancement of the early transient rise in [Ca 2+] i stimulated by PTH. PGF 2α was approximately 100 times more potent than PGE 2. PGE 2 itself was more potent than 6-keto-PGF 1α in enhancing PTH-stimulated rise in [Ca 2+] i. Near-maximal augmentation was achieved at PGF 2α doses of 10 nM and PGE 2 of 1 μM. The degree of augmentation in [Ca 2+] i by PGF 2α was independent of preincubation time. PGF 2α pretreatment did not alter the EC 50 for the PTH-induced [Ca 2+] i increase but only the extent of rise in [Ca 2+] i at each dose of PTH. The augmented increase in [Ca 2+] i was mostly due to enhanced PTH-mediated release of Ca 2+ from intracellular stores. PGF 2α did not stimulate an increase in PTH receptor number as assessed by [ 125I]-PTH-related peptide binding. PG pretreatment partially reversed PTH inhibition of cell proliferation, suggesting that an increase in [Ca 2+] i may play a role in tempering the anti-proliferative effect of PTH mediated by cAMP. These studies suggest a new mode by which PGs can affect cellular activity.