cAMP Phosphodiesterase Inhibitor Research Articles

Role of calcium-activated potassium channels and cyclic nucleotides on pulmonary vasoreactivity to serotonin.

The role of Ca(2+)-activated K+ channel modulation and cyclic nucleotide second messenger signal transduction in the canine pulmonary vascular response to serotonin was determined in the isolated blood-perfused dog lung. Pulmonary vascular resistances and compliances were measured using vascular occlusion techniques. Serotonin (10(-5) M) significantly increased precapillary and postcapillary resistance and significantly decreased total vascular compliance by decreasing large vessel compliance and middle compartment compliance. Tetraethylammonium ions (TEA+; 1 mM), an inhibitor of Ca(2+)-activated K+ channels, significantly potentiated the pressor effect to serotonin on both the pulmonary arteries and pulmonary veins. Pretreatment with the guanosine 3',5'-cyclic monophosphate (cGMP)/adenosine 3',5'-cyclic monophosphate (cAMP) phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (10(-5) M), the cell membrane-permeable analog of cAMP, dibutyryl-cAMP (10(-5) M), or the cAMP-dependent vasodilator isoproterenol (10(-5) M) inhibited the serotonergic response on both the arteries and veins, which was reversed by TEA+. In contrast, the stable membrane-permeable analog of cGMP, 8-bromo-cGMP (10(-5) M), had no effect on serotonin. These results indicate that there is a basal level of vasorelaxation in canine pulmonary blood vessels that is mediated by Ca(2+)-activated K+ channel activity and that inhibition of these K+ channels increases pulmonary vascular tone and potentiates the pulmonary vasoactive response to serotonin. Also, these data suggest that cAMP-induced pulmonary vasodilation is mediated primarily by Ca(2+)-activated K+ channels and that activation of these specific K+ channels attenuates the pressor response to serotonin. Thus an important relationship appears to exist between the cAMP second messenger system and Ca(2+)-activated K+ channels in canine pulmonary vasoreactivity.

Protein kinases selectively modulate apoptosis in the developing retina in vitro

In the retina of newborn rats there is evidence for two mechanisms of programmed cell death. Apoptosis of ganglion cells (RGCs) following axotomy depends on protein synthesis. In contrast, inhibition of protein synthesis leads to apoptosis in the neuroblastic layer (NBL). The induction of apoptosis following translational arrest suggests that post-translational modifications of apoptosis-associated proteins may be crucial to the cell death programs in the developing retina. We investigated the possible role of protein kinases upon apoptosis in retinal explants in vitro. An increase in the intracellular concentration of cAMP produced either by the adenylyl-cyclase activator forskolin (10 μM) or by 8-Br-cAMP (1 mM), prevented apoptosis induced in the NBL by inhibition of protein synthesis, but had no statistically significant effect upon RGC death. In contrast, neither 8-Br-cGMP (1 mM) nor the specific cGMP-phosphodiesterase inhibitor zaprinast (10–100 μM) had significant effects on apoptosis in the retina. The cAMP-phosphodiesterase inhibitors isobutylmethylxantine (IBMX, 0.1–1 mM) and Ro-201724 (50–200 μM) also prevented apoptosis in the NBL. The isoquinolinesulfonamide H89 (20 μM), a specific cAMP-dependent protein kinase inhibitor, partially reverted the protective effect of either forskolin or IBMX within the NBL. Neither 12-O-tetradecanoyl phorbol-13-acetate (TPA, 10 nM) nor bisindolylmaleimide (0.2–0.5 μM), respectively an activator and an inhibitor of protein kinase C had significant effects upon the retinal explants. The protein kinase inhibitor 2-aminopurine (2-AP, 10 mM) prevented apoptosis of axotomized ganglion cells and induced apoptosis in the NBL. Forskolin prevented the apoptosis induced by 2-AP in the NBL, whereas TPA had no effect. The effects of 2-AP were, however, not dependent on inhibition of protein synthesis. The data indicate that modulation of the activity of both cAMP-dependent protein kinase and several protein kinases sensitive to 2-aminopurine selectively affect apoptosis in distinct cell layers of the developing retina.

Proadrenomedullin NH2-terminal 20 peptide has direct vasodilator activity in the cat.

The mechanism by which proadrenomedullin NH2-terminal 20 peptide (PAMP) decreases vascular resistance was investigated in the hindlimb vascular bed in the cat. Injections of PAMP, a shortened form of the peptide PAMP-(12-20), and adrenomedullin (ADM) into the hindlimb perfusion circuit elicit dose-related decreases in perfusion pressure. The order of potency was ADM > PAMP > PAMP-(12-20), and the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP-(8-37) had no effect on vasodilator responses to PAMP or ADM. Vasodilator responses to PAMP were increased in duration by the adenosine 3',5'-cyclic monophosphate (cAMP) phosphodiesterase inhibitor Rolipram, whereas inhibitors of nitric oxide synthase and guanosine 3',5'-cyclic monophosphate phosphodiesterase had no effect. Vasodilator responses to PAMP were not altered by treatment with alpha-receptor or adrenergic nerve terminal blocking agents and were similar in innervated and denervated hindlimb preparations. Responses to PAMP were similar when vasoconstrictor tone was increased by stimulation of the sympathetic nerves or infusion of phenylephrine and were not altered by the passage of time. These data suggest that PAMP dilates the hindlimb vascular bed by a direct cAMP-dependent mechanism and that inhibition of norepinephrine release plays little if any role in mediating responses to the peptide in the regional vascular bed of the cat.

The effect of selective phosphodiesterase inhibitors on plasma insulin concentrations and insulin secretion in vitro in the rat

We have examined in rats the effects of Org 9935 (4,5-dihydro-6-(5,6-dimethoxy-benzo[ b]-thien-2-yl)-methyl-1-(2 H)-pyridazinone), a selective inhibitor of type 3 phosphodiesterase (phosphodiesterase 3) and Org 30029 ( N-hydroxy-5,6-dimethoxy-benzo[ b]-thiophene-2-carboximidamide HC1), an inhibitor of phosphodiesterase 3/4 on rat plasma insulin and glucose concentrations in pentobarbitone-anaesthetised rats and on insulin secretion by rat isolated islets. We have also compared their effects on islet phosphodiesterase activity. Org 9935 (0.1 and 1.0 mg kg −1 i.v. 15 min previously) dose dependently elevated fasting and post-glucose (0.25 g kg −1 i.v.) plasma insulin concentrations. Org 30029 in a dose of 10 mg kg −1, but not 1 mg kg −1, also increased plasma insulin concentrations. Neither drug modified either fasting or post-glucose plasma glucose concentrations. Each drug augmented glucose-induced insulin release by rat isolated islets in a static incubation system, with approximate EC 50 values of 1.5 μM for Org 9935 and 20 μM for Org 30029. Phosphodiesterase activity, in both supernatant and pellet fractions of islet homogenates, was inhibited concentration dependently by each drug. Although the shape of the concentration-inhibition curve for Org 30029 precluded estimation of an IC 50 value, this drug was clearly much less potent than Org 9935 (IC 50 about 50 nM) in inhibiting islet phosphodiesterase activity. We conclude that the increase in plasma insulin produced by each drug is a consequence of augmented insulin secretion, probably secondary to inhibition of phosphodiesterase 3 in the islet β cell, with a resultant elevation in cAMP. The failure of the drugs to modify plasma glucose may be due to concomitant inhibition of cAMP phosphodiesterase in liver and adipose tissue. © 1997 Elsevier Science B.V. All rights reserved.

Compartmentalization of cAMP Signaling in Mesangial Cells by Phosphodiesterase Isozymes PDE3 and PDE4 REGULATION OF SUPEROXIDATION AND MITOGENESIS

Some major pathobiologic processes in renal mesangial cells, elicited in response to immunoinflammatory stimuli, are modulated via cAMP-protein kinase A (PKA) signaling pathways; namely, generation of reactive oxygen metabolites (ROM) and accelerated proliferation of mesangial cells. We investigated the role of cAMP phosphodiesterase (PDE) isozymes in these regulatory mechanisms. Generation of ROM in cultured rat mesangial cells was inhibited by selective inhibitors of PDE4, rolipram and denbufylline, whereas PDE3 inhibitors, cilostamide and lixazinone, had no effect. Conversely, cilostamide or lixazinone suppressed mitogenic synthesis of DNA in mesangial cells, but 1 microM rolipram or 1 microM denbufylline showed no inhibitory effect. The efficacy of PDE isozyme inhibitors (IC50) to suppress [3H]thymidine incorporation or ROM generation paralleled IC50 values for inhibition of cAMP PDE. Incubation of mesangial cells with either rolipram alone or with cilostamide alone increased significantly in situ activity of PKA in mesangial cells, assessed by (-cAMP/+cAMP) PKA activity ratio, and the stimulatory effects were additive. Results indicate that in mesangial cells a cAMP pool that is metabolized by PDE4 activates PKA and thereby inhibits ROM generation; another cAMP pool that is metabolized by PDE3 activates another PKA (isozyme or pool) which suppresses proliferation of mesangial cells. We propose that in mesangial cells, a cAMP-PKA pathway that regulates mitogenesis is determined by activity of PDE3, whereas another cAMP-PKA pathway is directed by activity of PDE4 and controls ROM generation. Therefore, two PDE isozymes within one cell type compartmentalize distinct cAMP signaling pathways.

Chronic exposure to Ro20-1724 protects dopaminergic neurons in vitro against the neurotoxic action of N-methyl- d-aspartate and 1-methyl-4-phenylpyridinium

Using organotypic cultures of rat ventral mesencephalon, the effects of chronic (12–15 day) exposure to the type IV cAMP phosphodiesterase inhibitor, Ro20-1724, were examined. At concentrations of 10 −8–10 −5 M, Ro20-1724 alone had no effect upon the number of tyrosine hydroxylase-positive neurons or upon neurite outgrowth. However, the drug offered significant protection, with maximum effect at 10 −6 M, against subsequent acute (48 h) exposure to the neurotoxic agents 1-methyl-4-phenylpyridinium (MPP +) and N-methyl- d-aspartate (NMDA).

The roles of cGMP and cAMP in active thermoregulatory vasodilation.

This study was designed to test the hypothesis that active thermoregulatory vasodilation (AVD) is the result of a neurotransmitter-induced adenosine 3',5'-cyclic monophosphate (cAMP) pathway interacting with a nitric oxide-induced guanosine 3',5'-cyclic monophosphate (cGMP) pathway. Rabbits were instrumented for measurement of arterial pressure and ear blood flow (EBF) and the infusion of drugs. In four groups of conscious animals, whole-body heating increased EBF from 0.5 +/- 0.3 to 8.3 +/- 1.3 kHz. In group 1 (n = 6), N(omega)-nitro-L-arginine methyl ester (L-NAME, a nitric oxide synthase inhibitor, 10-40 mg) reduced EBF from 7.1 +/- 0.9 to 1.9 +/- 0.5 kHz. Subsequent infusion of 8-bromo-cGMP (a cGMP analog, 5-10 mg) returned EBF to 6.2 +/- 0.7 kHz. In group 2 (n = 3), (R)-p-adenosine 3',5'-cyclic monophosphothioate (a cAMP-dependent protein kinase inhibitor, 10 mg) reduced EBF to 1.6 +/- 0.4 kHz. In group 3 (n = 6), nerve blockade of the ear (procaine, 20 mg/ml, 1.5 ml) reduced EBF from 8.6 +/- 1.3 to 1.6 +/- 0.3 kHz. Subsequent infusion of 8-bromo-cAMP (a cAMP analog, 5-10 mg) returned EBF to 8.3 +/- 2.0 kHz. In group 4 (n = 6), the infusion of L-NAME caused EBF to fall from 9.0 +/- 1.1 to 1.2 +/- 0.3 kHz. Infusion of the cAMP phosphodiesterase inhibitor Ro 20-1724 (0.2-0.5 mg) raised EBF to 5.5 +/- 0.7 kHz. These results suggest that cGMP plays a permissive role in AVD and indicate that the transmitter acts through cAMP.

Stimulation or endothelin-1 secretion by human breast cancer cells through protein kinase A activation: a possible novel paracrine loop involving breast fibroblast-derived prostaglandin E 2

Breast cancer cells secrete endothelin-1 (ET-1), which may act as a paracrine mitogen in breast tumours. The paracrine factors and signal transduction pathways responsible for regulating ET-1 production in breast cancer are unknown. In this study we have examined the involvement of the protein kinase A (PKA) signalling pathway in the control of ET-1 secretion in the human breast cancer cell line MCF-7. Treatment of MCF-7 cells with various agents that activate protein kinase A (PKA) through increases in intracellular cAMP levels including forskolin, cholera toxin (ChT), the cAMP analogue 8-Br-cAMP, or the cAMP phosphodiesterase inhibitor, 3-isobutyl-1-methyl-xanthine (IBMX) all markedly increased ET-1 release. Prostaglandin E 2 (PGE 2) while stimulating cAMP production, but not inositol lipid hydrolysis also significantly stimulated ET-1 release. Activation of PKC by 12- O-tetradecanoyl phorbol 13-acetate (TPA) also stimulated ET-1 secretion in MCF-7 cells. The PKA inhibitor H-89 attenuated the ET-1 response to PGE 2, forskolin and ChT, but not that due to the PKC agonist TPA. The possibility that human breast fibroblasts (HBFs) are a target for ET-1 action with regard to PGE 2 production was also investigated, and revealed that while HBFs were unresponsive to ET-1 alone, pretreatment with the cytokine IL- β greatly potentiated PGE 2 release in response to ET-1. In conclusion our results show that activation of either the PKA or PKC signalling pathways in human breast cancer cells increases ET-1 secretion. We also found that HBFs release PGE 2 after treatment with ET-1 and that PGE 2 itself stimulates ET-1 production in MCF-7 cells. The implication of this potential novel paracrine loop may be significant in view of the high levels of PGE 2 and ET-1 found in malignant breast tissues.

Modulation of neuronal and glial cell function by adenosine and neuroprotection in vascular demential

Dementia is one of the major medical challenges in the industrialized countries. An increasing number of demented patients shows ischemic brain lesions. One strategy to treat dementia could be the reinforcement of the multifarious neuroprotective actions of the endogenous cell modulator adenosine. This involves the modulation of neuronal and glial cell functions. Adenosine appears to raise the threshold for the initiation of the pathophysiological cascade of ischemic neuronal death by counteracting ischemic membrane depolarization and the successive disturbance of intracellular Ca2+ homeostasis. When this threshold is overcome, adenosine seems to stimulate astrocyte differentiation and reinforce important protective astrocyte functions, e.g., synthesis and release of neurotrophic factors such as NGF and the clearance of abnormal neurotoxic levels of K+ and glutamate from the extracellular space. Propentofylline, a combined inhibitor of adenosine reuptake and cAMP phosphodiesterases, reinforces the actions of endogenous adenosine and cAMP. One important action of propentofylline is the inhibition of potentially neurotoxic functions of activated microglia (free radical formation and transformation into brain macrophages). Such drugs may help to inhibit the progressive neurodegenerative process in dementia.

Protein kinase A-mediated enhancement of miniature IPSC frequency by noradrenaline in rat cerebellar stellate cells.

1. Cellular mechanisms underlying the enhancement by noradrenaline (NA) of inhibitory postsynaptic currents (IPSCs) were studied at inhibitory synapses in the molecular layer of the cerebellum. IPSCs were obtained from stellate cells in rat cerebellar slices using tight-seal whole-cell recording. 2. Miniature IPSCs (mIPSCs) were recorded in the presence of tetrodotoxin (TTX; 100 nM). NA (10 microM) markedly increased the frequency of mIPSCs, but did not alter their mean amplitude. Bath application of the inhibitor of adenylyl cyclase 9-(tetrahydro-2'-furyl) adenine (SQ 22,536; 300 microM), of the wide spectrum protein kinase inhibitor staurosporine (1 microM), and of the Rp-diastereomer of adenosine-3',5'-cyclic monophosphothioate (Rp-cAMPS; 500 microM), a specific inhibitor of cAMP-dependent protein kinase (PKA), inhibited the mIPSC frequency increase induced by NA. 3. The increase in mIPSC frequency was not attenuated by Cd2+ (100 microM), a blocker of voltage dependent calcium channels. However, after a 12-15 min pre-incubation in Ca(2+)-free saline, the effect of NA on mIPSCs was markedly inhibited. If Ca2+ ions were readmitted in the presence of NA, enhancement of the mIPSC frequency was largely restored. 4. Application of the membrane permeant analogue of cAMP, 8-Br-cAMP (1 mM), together with the inhibitor of cAMP phosphodiesterase, 3-isobutyl-1-methylxanthine (IBMX; 100 microM), caused a frequency increase of mIPSCs. Forskolin also mimicked the stimulatory effect of NA on mIPSC frequency. The effects of both 8-Br-cAMP and forskolin persisted in Ca(2+)-free saline, suggesting that the modulation of transmitter release does not require Ca2+ influx. 5. On the whole, the results indicate that the potentiation of mIPSC frequency by NA is mediated through the sequential activation of adenylyl cyclase and protein kinase A (PKA), and that PKA modulates the vesicle release mechanism rather than Ca2+ influx. The lack of effect of NA after prolonged incubation in Ca(2+)-free solution may be due to an inhibition of adenylyl cyclase by a gradual lowering of the cytosolic presynaptic Ca2+ concentration.

Specific inhibition of the cGMP-inhibited cAMP phosphodiesterase blocks the insulin-like antilipolytic effect of growth hormone in rat adipocytes.

Growth hormone (GH) exerts acute insulin-like effects, such as increased lipogenesis and inhibition of catecholamine-induced lipolysis, in rat adipocytes that have not been exposed to GH during the preceding three hours. We found that OPC3911, a highly specific inhibitor of the cGMP-inhibited cAMP phosphodiesterase, completely blocked the antilipolytic but not the lipogenic effect of GH. This indicates that the antilipolytic effect of GH is mediated through activation of this phosphodiesterase leading to reduction of cAMP levels in the same manner as has been shown for insulin.

Adrenomedullin-(22-52) antagonizes vasodilator responses to CGRP but not adrenomedullin in the cat.

The effects of adrenomedullin (ADM)-(22-52), a putative ADM receptor antagonist, on vasodilator responses to ADM and the structurally related peptide, calcitonin gene-related peptide (CGRP), were investigated in the hindlimb vascular bed of the cat under constant-flow conditions. ADM-(22-52) had no significant effect on hindlimb perfusion pressure when injected in doses up to 120 nmol; after administration of ADM-(22-52), vasodilator responses to ADM were unchanged, whereas vasodilator responses to CGRP were inhibited. The inhibitory effects of ADM-(22-52) on responses to CGRP were selective and reversible and were similar to the inhibitory effects of the CGRP antagonist CGRP-(8-37). Hindlimb vasodilator responses to CGRP and to ADM were increased in duration by the adenosine 3',5'-cyclic monophosphate (cAMP) phosphodiesterase inhibitor rolipram but were not altered by inhibitors of guanosine 3',5'-cyclic monophosphate phosphodiesterase, nitric oxide synthetase, K(+)-ATP channels, the cyclooxygenase pathway, or the adrenergic nervous system. These results demonstrate that ADM-(22-52) is a selective CGRP receptor antagonist in the hindlimb vascular bed of the cat. The present data suggest that vasodilator responses to CGRP and ADM are mediated by different receptors but that these peptides dilate the hindlimb vascular bed of the cat by a similar cAMP-dependent mechanism.

Cardiotonic activity of the rhizome of Polygonatum sibiricum in rats.

The cardiotonic effect of the rhizome of Polygonatum sibiricum was investigated in the left atria of rats. The methanol extract of the rhizome of Polygonatum sibiricum (OM) (1-7 mg/ml) concentration-dependently increased the developed tension of the left atrium. It also strongly inhibited cAMP phosphodiesterase. The increase cAMP level correlated the increase in left atrial contraction. On the other hand, OM did not inhibit Na+, K+-ATPase. The cardiotonic effect of OM was strongly inhibited by reserpine, a sympatholytic agent. Furthermore, OM-treated left atria inhibited the tension produced by propranolol, a beta adrenocepter antagonist. These findings suggested that the cardiotonic effect is due to stimulating beta adrenoceptors through activation of sympathetic nerves.

Screening of Some Marine Organism Extracts for cAMP Phosphodiesterase Inhibition, Cytotoxicity, and Antiviral Activity against HSV-1

Methanol and hot water extracts of 71 samples of 56 kinds of marine products were subjected to inhibitory test for cyclicAMP phosphodiesterase (cAMP PDE), cytotoxic and anti-viral screening tests. Among them, 7 extracts of marine products showed a significant inhibition for cAMP PDE and 5 extracts showed a significant cytotoxicity against KB cells at concentrations under 20 µg/ml. Eight extracts of marine products showed higher therapeutic indexes against herpes simplex virus type 1 (HSV-1).

Modulation of the Antiviral 2-5A System in Human Immunodeficiency Virus-1-Infected CEM Cells by Propentofylline

2′,5′-OIigoadenylates (2-5A) play an essential role in the establishment of the antiviral state of cells exposed to virus infection. However, - after an initial increase observed in some cell lines - the activity of the interferon (IFN)-inducible, 2-5A-forming 2′,5′-oligoadenylate synthetase (2-5A synthetase) strongly decreases soon after infection of cells with the human immunodeficiency virus-1 (HIV-1). In the present report, we show that in IFN-treated human T lymphoblastoid CEM cells, the decrease in 2-5A synthetase activity had already occurred at day 1 post infection (p.i.)- At days 3 and 5 p.i., the 2-5A synthetase activity in the IFN-treated infected cells amounted to only 10-12% of that in IFN-treated uninfected control cells. The decrease in 2-5A synthetase activity was accompanied by a decrease in 2-5A synthetase mRNA and protein. We found that the decrease in 2-5A synthetase activity can be retarded by addition of the cAMP phosphodiesterase inhibitor, propentofylline. At a concentration of 30-100 μM, propentofylline displayed a significant cytoprotective and antiviral effect on HIV-1-infected CEM cells.

Effect of cilostazol, a cAMP phosphodiesterase inhibitor, on nitric oxide production by vascular smooth muscle cells

We investigated the effects of cilostazol, a cAMP phosphodiesterase inhibitor, on nitric oxide (NO) synthesis in cultured rat vascular smooth muscle cells. Incubation of the cultures with interleukin-1β (10 ng/ml) for 24 h caused a significant increase in the accumulation of nitrite, a stable metabolite of NO. Although cilostazol by itself showed no effect on nitrite accumulation, it stimulated interleukin-1β-induced nitrite accumulation in a concentration-dependent manner (10 −8–10 −5 M). This effect of cilostazol was completely abolished in the presence of N G-monomethyl- l-arginine, actinomycin D or dexamethasone. The cilostazol-induced nitrite production was accompanied by increased inducible NO synthase protein expression. In the presence of dibutyryl-cAMP, interleukin-1β-induced nitrite accumulation was further increased, but the stimulatory effect of cilostazol on nitrite accumulation was blunted. The effect of cilostazol was also abolished in the presence of Rp-8-bromoadenosine-3′,5′-cyclic monophosphorothioate, a competitive inhibitor of protein kinase A. Addition of cilostazol to the cultures significantly increased intracellular cAMP levels of vascular smooth muscle cells. These results indicate that cilostazol increases NO synthesis in interleukin-1β-stimulated vascular smooth muscle cells, at least partially through a cAMP-dependent pathway.

The Bioactive Phospholipid, Lysophosphatidylcholine, Induces Cellular Effects via G-Protein-dependent Activation of Adenylyl Cyclase

The naturally occurring phospholipid, lysophosphatidylcholine (lyso-PC), regulates a broad range of cell processes, including gene transcription, mitogenesis, monocyte chemotaxis, smooth muscle relaxation, and platelet activation. Despite the growing list of cellular effects attributable to lyso-PC, the mechanism(s) by which it alters cell function have not been elucidated. In this report, we have examined the effects of exogenous lyso-PC on signal transduction processes within a variety of lyso-PC-responsive cells, including human platelets, monocyte-like THP-1 cells, and the megakaryoblastic cell line, MEG-01. Pretreatment of each of these cells with increasing concentrations of lyso-PC (25-150 microg/ml) was associated with a progressive increase in the cytosolic concentration of cAMP. The accumulation of cAMP in platelets correlated closely with the ability of lyso-PC to inhibit multiple platelet processes, including platelet aggregation, agonist-induced protein kinase C activation, thromboxane A2 generation, and the tyrosine phosphorylation of platelet proteins. In each of the cell types examined, the ability of lyso-PC to increase the cellular levels of cAMP was synergistically enhanced by pretreating the cells with the cAMP phosphodiesterase inhibitor, theophylline (5 mM), and was specifically inhibited by the P-site inhibitor of adenylyl cyclase, 2,5-dideoxyadenosine. A role for the stimulatory G-protein, Gs, in the lyso-PC-induced activation of adenylyl cyclase was suggested by the ability of the GTPase inhibitor, guanylyl 5'-thiophosphate (0.2 mM), to inhibit the lyso-PC-stimulated increase in cAMP, and also by the ability of cholera toxin to inhibit increases in membrane GTPase activity in response to lyso-PC. The functional significance of lyso-PC-induced activation of adenylyl cyclase was investigated in MEG-01 cells. Treatment of these cells with either lyso-PC or dibutyryl cAMP for 36-40 h resulted in a 3-5-fold increase in the surface expression of the natural anticoagulant protein, thrombomodulin (TM). The ability of lyso-PC to increase TM expression was abolished by pretreating these cells with the adenylyl cyclase inhibitor, 2,5-dideoxyadenosine, whereas the dibutyryl cAMP-induced increase in TM remained insensitive to adenylyl cyclase inhibition. These studies define an important role for the adenylyl cyclase signaling system in mediating cellular effects induced by lyso-PC.

Transcriptional enhancement of tyrosine hydroxylase by prostaglandin E2 in SK-N-BE(2) C cells.

Transcriptional regulation of the rat tyrosine hydroxylase (TH) gene by prostaglandin E2 (PGE2) was investigated in human neuroblastoma SK-N-BE(2)C cells. Prostaglandins increased intracellular cAMP in the presence of 3-isobutyl-1-methylxanthine (IBMX), a cAMP phosphodiesterase inhibitor. Among the prostaglandins tested for their cAMP raising property PGE2 was the most effective. The results suggest that the cells express adenylyl cyclase-linked prostanoid receptors that have a higher affinity for PGE2 than for any other naturally occurring prostaglandin. The treatment of cells with PGE2 increased the TH gene expression approximately 2-fold, even though the cAMP accumulation induced by PGE2 alone was almost negligible. Simultaneous treatment with PGE2 and IBMX enhanced the gene expression concomitantly with a marked accumulation of cAMP. Transient transfection assays with 5' upstream serially deleted constructs of the rat TH gene promoter region fused to the chloramphenicol acetyltransferase (CAT) gene revealed that a cAMP response element (CRE) located at -45 to -38 from the start of the TH gene was essential for the enhancement of TH gene expression by PGE2. Site-directed mutagenesis and specific deletion within the sequence of the CRE motif abolished the transcriptional enhancement by PGE2. In addition, a protein kinase A (PKA) inhibitor, H89, specifically blocked the PGE2 effect on TH gene expression. Northern blot analysis revealed that the increase in TH gene transcription with PGE2 is associated with an elevated TH mRNA level. Gel retardation and competition assays confirmed that the binding of nuclear factors to the CRE site was sequence specific and was augmented by PGE2. Our data indicate that PGE2 enhances transcription of the TH gene mediated by the CRE motif through the activation of PKA. They also suggest that the signal flow from the adenylyl cyclase-linked prostanoid receptor to the nucleus is efficient although cAMP accumulation is not prominent.

Effect in vitro of gamma interferon and interleukin-10 on generation of oxidizing species by human granulocytes.

We examined the effect of interleukin-10 (IL-10), gamma interferon (IFN-gamma), phorbol ester (PDB), opsonized zymosan (OZ) and aminophylline (a cAMP phosphodiesterase inhibitor) on the reducing power and oxidizing species generation by human neutrophils, using MTT dye reduction and luminol-dependent chemiluminescence assays, respectively. Gamma interferon (IFN-gamma), phorbol ester (PDB) and opsonized zymosan (OZ) were activators while interleukin-10 (IL-10) and aminophylline were inhibitors. A strong parallelism was observed between oxidizing species generation and cellular reducing power in both activation and inhibition experiments. Our results also demonstrate for the first time the effect of IL-10 on free radical generation by neutrophils. The consequence of these activating and inhibiting effects on the inflammatory process are discussed.

Nitric oxide synthase (NOS3) and contractile responsiveness to adrenergic and cholinergic agonists in the heart. Regulation of NOS3 transcription in vitro and in vivo by cyclic adenosine monophosphate in rat cardiac myocytes.

Cardiac myocytes express the nitric oxide synthase isoform originally identified in constitutive nitric oxide synthase cells (NOS3), which mediates the attenuation by muscarinic cholinergic agonists of beta-adrenergic stimulation of L-type calcium current and contractility in these cells. However, calcium current and contractility in these cells. However, the reciprocal regulation of NOS3 activity in myocytes by agents that elevate cAMP has not been reported. In this study, we show that NOS3 and mRNA and protein levels in cardiac myocytes are reduced both in vitro after treatment with cAMP elevating drugs, and in vivo after 3 d of treatment with milrinone, a type III cAMP phosphodiesterase inhibitor. This effect on NOS3 activity by cAMP is cell type specific because treatment of cardiac microvascular endothelial cells in vitro or in vivo did not decrease NOS3 mRNA or protein in these cells. NOS3 downregulation in myocytes appeared to be at the level of transcription since there was no modification of NOS3 mRNA half-life by agents that increase intracellular cAMP. To determine the functional effects of NOS3 downregulation, we examined the contractile responsiveness of isolated electrically paced ventricular myocytes, isolated from animals that had been treated in vivo with milrinone, to the beta-adrenergic agonist isoproterenol and the muscarinic cholinergic agonist carbamylcholine. There was no difference in baseline contractile function in cells that had been pretreated with cAMP elevating agents compared to controls, but cells exposed to milrinone in vivo exhibited an accentuation in their contractile responsiveness to isoproterenol compared to controls and a loss of responsiveness to carbamylcholine. Downregulation of myocyte NOS3 by sustained elevation of cAMP may have important implications for the regulation of myocardial contractile state by the autonomic nervous system.