Cyclic AMP Phosphodiesterase Activity Research Articles

Critical evaluation of food and mite allergy in the management of atopic dermatitis.

The subject of allergic causation of atopic dermatitis (AD) has been a source of controversy for over sixty years. While there is general agreement of allergen-induced asthma and allergic rhinoconjunctivitis, AD lesions cannot be readily or consistently induced by foods and inhalants. The most consistent perturbators of atopic conditions are irritants, probably reflecting the consistent inflammatory cell hyper-reactivity due to elevated cyclic AMP-phosphodiesterase activity. The immunologic reactions are another manifestation of that general cellular dysregulation.

Type 4 Phosphodiesterase Inhibitors Have Clinical and In Vitro Anti-inflammatory Effects in Atopic Dermatitis

Increased cyclic AMP-phosphodiesterase activity in peripheral blood leukocytes is associated with the immune and inflammatory hyperreactivity that characterizes atopic dermatitis. Atopic phosphodiesterase has high sensitivity to a variety of enzyme inhibitors, suggesting an increased therapeutic advantage. The objective of this study was to use in vitro assays to identify a potent phosphodiesterase inhibitor and then to investigate its effectiveness in treating atopic dermatitis. Leukocyte enzyme activity was measured by radioenzyme assay, whereas prostaglandin E2 and interleukins 10 (IL-10) and 4 (IL-4) were measured in 24-h culture supernatants of mononuclear leukocytes by immunoassays. The effect of a topical phosphodiesterase inhibitor on atopic dermatitis lesional skin was assessed by double-blind, paired comparisons of active drug and placebo ointments applied to symmetrically involved sites over a 28-d period. Using in vitro, assays, we demonstrated the ability of selective high-potency phosphodiesterase inhibitors to reduce prostaglandin E2, IL-10, and IL-4 production in atopic mononuclear leukocyte cultures. We selected the Type 4 phosphodiesterase inhibitor, CP80,633, based on its inhibitory potency, for clinical testing by topical, bilateral paired comparisons in 20 patients with atopic dermatitis and demonstrated significant reductions of all inflammatory parameters. Phosphodiesterase inhibitors modulate several pathways contributing to the exaggerated immune and inflammatory responses, which characterize atopic dermatitis. This in vivo demonstration of anti-inflammatory efficacy may provide a useful alternative to the over-reliance on corticosteroid therapy in atopic disease.

Rapid regulation of PDE-2 and PDE-4 cyclic AMP phosphodiesterase activity following ligation of the T cell antigen receptor on thymocytes: Analysis using the selective inhibitors erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA) and rolipram

The PDE2, cyclic GMP-stimulated, and the PDE4, cyclic AMP-specific enzymes provide the major, detectable cyclic AMP phosphodiesterase activities in murine thymocytes. In the absence of cyclic GMP, PDE4 activity predominated (∼80% total) but in the presence of low (10 μM) cyclic GMP concentrations, PDE2 activity constituted the major PDE activity in thymocytes (∼80% total). The PDE4 selective inhibitor rolipram dose-dependently inhibited thymocyte PDE4 activity ( ic 50 ∼ 65 nM). PDE2 was dose-dependently activated ( ec 50 ∼ 1 μM) by cyclic GMP and inhibited by erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA) ( ic 50 ∼ 4 μM). EHNA was shown to serve as a selective inhibitor of PDE-2 activity as assessed from studies using separated PDE1, PDE2, PDE3 and PDE4 species from hepatocytes as well as human PDE2 and PDE4 enzymes. EHNA completely ablated the ability of cyclic GMP to activate PDE2 activity, whilst having a much smaller inhibitory effect on the unstimulated PDE2 activity. EHNA exhibited normal Michaelian kinetics of inhibition for the cyclic GMP-stimulated PDE2 activity with Hill plots near unity. Apparent negative co-operative effects were seen in the absence of cyclic GMP with Hill coefficients of ∼0.3 for inhibition of PDE2 activity. Within 5 min of challenge of thymocytes with the lectin phytohaemagglutinin (PHA) there was a transient decrease (∼83%) in PDE-4 activity and in PDE2 activity (∼40%). Both anti-CD3 and anti-TCR antibodies also caused an initial reduction in the PDE4 activity which was followed by a sustained and profound increase in activity. In contrast to that observed with PHA, anti-TCR/CD3 antisera had little effect on PDE2 activity. It is suggested that, dependent upon the intracellular concentrations of cyclic GMP, thymocyte cyclic AMP metabolism can be expected to switch from being under the predominant control of PDE4 activity to that determined predominantly by PDE2 activity. These activities may be rapidly and differentially regulated following ligation of different cell surface receptors.

Therapeutic actions of garlic constituents.

Most studies on garlic during the past 15 years have been primarily in the fields of cardiovascular and cancer research. Cardiovascular studies have been mainly related to atherosclerosis, where effects were examined on serum cholesterol, LDL, HDL, and triglycerides. Although the studies were not consistent in relation to the dosage, standardization of garlic preparations, and period of treatment, most findings suggest that garlic decreases cholesterol and triglycerides levels in patients with increased levels of these lipids. Lowering of serum lipids by garlic ingestion may decrease the atherosclerosis process. The other major beneficial effect of garlic is due to its antithrombotic actions. This field of garlic research has been extensively studied. Garlic extracts and several garlic constituents demonstrate significant antithrombotic actions both in vitro and in vivo systems. Allicin and adenosine are the most potent antiplatelet constituents of garlic because of their in vitro effects. Since both allicin and adenosine are rapidly metabolized in human blood and other tissues, it is doubtful that these compounds contribute to any antithrombotic actions in the body. In addition, ajoene also seems not to be an active antiplatelet principle, because it is not naturally present in garlic, garlic powders, or other commercial garlic preparations. Only a small amount of ajoene can be found in garlic oil-macerates; however, ajoene is being developed as a drug for treatment of thromboembolic disorders. Recent findings on the identification of potent enzyme inhibiting activities of adenosine deaminase and cyclic AMP phosphodiesterase in garlic extracts are interesting, and may have a significant role in the pharmacological actions in the body. Presence of such enzyme inhibitors in garlic may perhaps explain several clinical effects in the body, including the antithrombotic, vasodilatory, and anticancer actions. Epidemiological studies have suggested that garlic plays a significant role in the reduction of deaths caused by malignant diseases. This had led many investigators to examine garlic and garlic constituents for their antitumor and cytotoxic actions both in vitro and in laboratory animals. The data from these investigations suggest that garlic contains several potentially important agents that possess antitumor and anticarcinogenic properties. In summary, the epidemiological, clinical, and laboratory data have proved that garlic contains many biologically and pharmacologically important compounds, which are beneficial to human health from cardiovascular, neoplastic, and several other diseases. Numerous studies are in progress all over the world to develop effective and odorless garlic preparations, as well as to isolate the active principles that may be therapeutically useful.

A novel phosphodiesterase inhibitor, T-440: possible management of eosinophilic inflammation by down-regulation of interleukin-5 production.

The effect of T-440, a selective type IV phosphodiesterase (PDE IV) inhibitor on interleukin (IL)-5 production by peripheral blood mononuclear cells (PBMCs) of atopic asthmatic subjects was investigated. PBMCs produced IL 5 following challenge with specific allergen in vitro. T-440 suppressed allergen-induced IL-5 production significantly at a concentration of 1 microgram/ml. T-440 inhibited cyclic AMP-phosphodiesterase (PDE) activity in a concentration dependent manner and raised the intracellular cyclic AMP level of PBMCs significantly. Dibutyryl cyclic AMP suppressed IL-5 production by PBMCs in a similar way to T-440, suggesting that the increase of intracellular cyclic AMP caused by T-440 reduces IL-5 production. T-440 may be an effective agent to treat atopic diseases associated with eosinophilic inflammation, e.g. asthma and atopic dermatitis.

Cross-talk between glucagon- and adenosine-mediated signalling systems in rat hepatocytes: effects on cyclic AMP-phosphodiesterase activity.

The effect of adenosine analogues on glucagon-stimulated cyclic AMP accumulation in rat hepatocytes was explored. N6-Cyclopentyladenosine (CPA), 5'-N-ethylcarboxamidoadenosine and N6-(R-phenylisopropyl)adenosine inhibited in a dose-dependent manner the cyclic AMP accumulation induced by glucagon. This effect seems to be mediated through A1 adenosine receptors. Pertussis toxin completely abolished the effect of CPA on glucagon-stimulated cyclic AMP accumulation in whole cells which suggested that a pertussis-toxin-sensitive G-protein was involved. On the other hand, this action of adenosine analogues on glucagon-induced cyclic AMP accumulation was reverted by the selective low-Km cyclic AMP-phosphodiesterase inhibitor Ro 20-1724. Analysis of cyclic AMP-phosphodiesterase activity in purified hepatocyte plasma membranes showed that glucagon in the presence of GTP inhibited basal PDE activity by 45% and that CPA reverted this inhibition in dose-dependent manner. In membranes derived from pertussis-toxin-treated rats, we observed no inhibition of cyclic AMP-phosphodiesterase activity by glucagon in the absence or presence of CPA. Our results indicate that in hepatocyte plasma membranes, stimulation of adenylate cyclase activity and inhibition of a low-Km cyclic AMP phosphodiesterase activity are co-ordinately regulated by glucagon, and that A1 adenosine receptors can inhibit glucagon-stimulated cyclic AMP accumulation by blocking glucagon's effect on phosphodiesterase activity.

Induction of Ca2+/calmodulin-stimulated cyclic AMP phosphodiesterase (PDE1) activity in Chinese hamster ovary cells (CHO) by phorbol 12-myristate 13-acetate and by the selective overexpression of protein kinase C isoforms

The cAMP phosphodiesterase (PDE) activity of CHO cells was unaffected by the addition of Ca2+ +calmodulin (CaM), indicating the absence of any PDE1 (Ca2+/CaM-stimulated PDE) activity. Treatment with the tumour promoting phorbol ester phorbol 12-myristate 13-acetate (PMA) led to the rapid transient induction of PDE1 activity which attained a maximum value after about 13 h before slowly decreasing. Such induction was attenuated by actinomycin D. PCR primers were designed to hybridize with two regions identified as being characteristic of PDE1 forms found in various species and predicted to amplify a 601 bp fragment. RT-PCR using degenerate primers allowed an approx. 600 bp fragment to be amplified from RNA preparations of rat brain but not from CHO cells unless they had been treated with PMA. CHO cells transfected to overexpress protein kinase C (PKC)-alpha and PKC-epsilon, but not those transfected to overexpress PKC-beta I or PKC-gamma, exhibited a twofold higher PDE activity. They also expressed a PDE1 activity, with Ca2+/CaM effecting a 1.8-2.8-fold increase in total PDE activity. RT-PCR, with PDE1-specific primers, identified an approx. 600 bp product in CHO cells transfected to overexpress PKC-alpha and PKC-epsilon, but not in those overexpressing PKC-beta I or PKC-gamma. Treatment of PKC-alpha transfected cells with PMA caused a rapid, albeit transient, increase in PDE1 activity, which reached a maximum some 1 h after PMA challenge, before returning to resting levels some 2 h later. The residual isobutylmethylxanthine (IBMX)-insensitive PDE activity was dramatically reduced (approx. 4-fold) in the PKC-gamma transfectants, suggesting that the activity of the cyclic AMP-specific IBMX-insensitive PDE7 activity was selectively reduced by overexpression of this particular PKC isoform. These data identify a novel point of 'cross-talk' between the lipid and cyclic AMP signalling systems where the action of specific PKC isoforms is shown to cause the induction of Ca2+/CaM-stimulated PDE (PDE1) activity. It is suggested that this protein kinase C-mediated process might involve regulation of PDE1 gene expression by the AP-1 (fos/jun) system.

Effects of type‐selective phosphodiesterase inhibitors on glucose‐induced insulin secretion and islet phosphodiesterase activity

1. We examined various type-selective phosphodiesterase (PDE) inhibitors on glucose-induced insulin secretion from rat isolated islets, on islet PDE activity and on islet cyclic AMP accumulation in order to assess the relationship between type-selective PDE inhibition and modification of insulin release. 2. The non-selective PDE inhibitor, 3-isobutyl-1-methylxanthine (IBMX, 10(-5)-10(-3) M), as well as the type III selective PDE inhibitors SK&F 94836 (10(-5)-10(-3) M), Org 9935 (10(-7)-10(-4) M), SK&F 94120 (10(-5)-10(-4) M) and ICI 118233 (10(-6)-10(-4) M) each caused concentration-dependent augmentation (up to 40% increase) of insulin release in the presence of a stimulatory glucose concentration (10 mM), but not in the presence of 3 mM glucose. 3. Neither the type IV PDE inhibitor rolipram (10(-4) M) nor the type I and type V PDE inhibitor, zaprinast (10(-4)-10(-3) M) modified glucose-induced insulin release when incubated with islets, although a higher concentration of rolipram (10(-3) M) inhibited secretion by 55%. However, when islets were preincubated with these drugs followed by incubation in their continued presence, zaprinast (10(-6)-10(-4) M) produced a concentration-dependent inhibition (up to 45% at 10(-4) M). Under these conditions, rolipram inhibited insulin secretion at a lower concentration (10(-4) M) than when simply incubated with islets. 4. A combination of SK&F 94836 (10(-5) M) and forskolin (5 x 10(-8) M) significantly augmented glucose-induced insulin secretion (30% increase), although neither drug alone, in these concentrations, produced any significant effect. 5. Islet cyclic AMP levels, which were not modified by forskolin (10-6 M), SK&F 94836 (10-4 M) or Org 9935 (10-5 M) were significantly elevated (approximately 3.7 fold increase) by forskolin inc ombination with either SK&F 94836 or Org 9935.6 Homogenates of rat islets showed a low Km (1.7 microM) and high Km (13 microM) cyclic AMP PDE in the supernatant fractions (from 48,000 g centrifugation), whereas the particulate fraction showed only a low Km (1.4 microM) cyclic AMP PDE activity.7. The PDE activity of both supernatant and pellet fractions were consistently inhibited by SK&F94836 or Org 9935, the concentrations required to reduce particulate PDE activity by 50% being 5.5 and 0.05 microM respectively.8 Rolipram (10-5 10-4 M) did not consistently inhibit PDE activity in homogenates of rat islets and zaprinast (10-4 M) consistently inhibited activity by 30% in the supernatant fraction, but not consistently in the pellet.9 These data are consistent with the presence of a type III PDE in rat islets of Langerhans.

Insulin-Stimulated Calmodulin Gene Expression in Rat H-411E Cells Can Be Selectively Blocked by Antisense Oligonucleotides

Reduced expression of calmodulin (CaM) and decreased activity of low Km cyclic AMP (cAMP) phosphodiesterase (PDE) are associated with uncontrolled diabetes. This condition can be readily mimicked in hepatocytes cultivated in insulin-depleted medium (Solomon, et al J. Lab. Clin. Med. in press, 1994). To investigate the relationship between CaM and low Km cAMP PDE gene expression in response to insulin, we specifically blocked expression of the three CaM genes by antisense oligonucleotides under insulin-deficient and -sufficient conditions in a rat hepatoma cell line, H-411E. We observed that both the low Km cAMP PDE activity and the steady state levels of CaM mRNA were increased in response to insulin by 50 and 100%, respectively. When antisense oligonucleotide to CaM I, II or III was added to the cultures, only CaM I antisense oligonucleotide blocked insulin stimulation of both CaM I mRNA and protein with concommittant marked inhibition of insulin′s expected stimulation of low Km cAMP PDE. Furthermore, in another experiment utilizing both antisense and oligonucleotide probes specific for CaM I,II, or III together, only CaM I mRNA expression was blocked. We conclude that H-411E cells respond to insulin by appropriate increases in CaM transcripts. Furthermore, the stimulatory effect of insulin on both CaM synthesis and activation of low Km cAMP PDE could be blocked by antisense to CaM I, but not II or III genes. Therefore, in addition to the above conclusions, H-411E hepatoma cells appear to be an excellent in vitro system to explore the molecular mechanisms by which CaM and low Km cAMP PDE genes are regulated in the diabetic state.

Effects of chemical denervation with 6-hydroxydopamine on myocardial responsiveness to isoproterenol in rabbits.

This study examined the hypothesis that chemical denervation with 6-hydroxydopamine (6-OHDA) would increase myocardial responsiveness to isoproterenol. Five days previously, 15 New Zealand white rabbits were given 60 mg/kg 6-OHDA intravenously. Fifteen control rabbits received vehicle. Hemodynamic, coronary blood flow (CBF), and cardiac output measurements were obtained before and during isoproterenol infusion (0.5 microgram/kg/min for 15 min). Norepinephrine tissue content, beta-adrenoceptor number (Bmax) and affinity (Kd), cyclic AMP content and cyclic AMP-phosphodiesterase (PDE) activity were measured in the subepicardium (EPI) and subendocardium (ENDO). Myocardial norepinephrine content was significantly decreased from 1263 +/- 292 (EPI) and 874 +/- 221 ng/g tissue (ENDO) in the control to 148 +/- 33 (EPI) and 90 +/- 45 ng/g tissue (ENDO) in the denervated group. There were no significant changes in cyclic AMP-PDE activity or Bmax and Kd of beta-adrenoceptors. Cyclic AMP content was similar at baseline, but controls had a significantly larger increase (123-155%) during isoproterenol infusion when compared to the denervated group (27-37%). The denervated animals showed a smaller increase in cardiac output during isoproterenol infusion (from 203 +/- 30 to 235 +/- 26 ml/min), when compared to the control animals (from 135 +/- 18 to 216 +/- 42 ml/min). Baseline CBF was significantly higher in the EPI but not ENDO of the denervated group (185 +/- 20 ml/100 g/min in EPI and 150 +/- 8 in ENDO) compared to the control group (108 +/- 13 in EPI and 133 +/- 17 in ENDO). The relative increase in CBF during isoproterenol infusion was smaller in the denervated group (44-45%) than the control group (107-109%). Isoproterenol infusions of 0.1 and 2.5 micrograms/kg/min showed similarly depressed coronary blood flow responses in denervated animals. Thus, the chemically denervated animals did not have beta-adrenoceptor upregulation, exhibited a lesser increase in cyclic AMP with isoproterenol, and had a reduced functional and coronary blood flow response to isoproterenol. This occurred without any significant change in beta-adrenoceptor number or affinity, or in cyclic AMP-phosphodiesterase activity, indicating there may be receptor uncoupling or other changes in the signal transduction pathway.

Further analysis of the mechanisms underlying the tracheal relaxant action of SCA40.

1. SCA40 (1nM-10 microM), isoprenaline (1-300 nM) and levcromakalim (100 nM-10 microM) each produced concentration-dependent suppression of the spontaneous tone of guinea-pig isolated trachea. Propranolol (1 microM) markedly (approximately 150 fold) antagonized isoprenaline but did not antagonize SCA40. The tracheal relaxant action of SCA40 was unaffected by suramin (100 microM) or 8-(p)-sulphophenyltheophylline (8-SPT; 140 microM). 2. An isosmolar, K(+)-rich (80 mM) Krebs solution increased tracheal tone, antagonized SCA40 (approximately 60 fold), antagonized isoprenaline (approximately 20 fold) and very profoundly depressed the log concentration-effect curve for levcromakalim. Nifedipine (1 microM) did not itself modify the relaxant actions of SCA40, isoprenaline or levcromakalim. However, nifedipine prevented the rise in tissue tone and the antagonism of SCA40 and isoprenaline induced by the K(+)-rich medium. In contrast, nifedipine did not prevent the equivalent antagonism of levcromakalim. 3. Charybdotoxin (100 nM) increased tracheal tone, antagonized SCA40 (approximately 4 fold) and antagonized isoprenaline (approximately 3 fold). Nifedipine (1 microM) prevented the rise in tissue tone and the antagonism of SCA40 and isoprenaline induced by charybdotoxin. 4. Quinine (30 microM) caused little or no change in tissue tone and did not modify the relaxant action of isoprenaline. However, quinine antagonized SCA40 (approximately 2 fold). Nifedipine (1 microM) prevented the antagonism of SCA40 induced by quinine. 5. Tested on spontaneously-beating guinea-pig isolated atria SCA40 (1 nM-10 microM) increased the rate of beating in a concentration-dependent manner. Over the concentration-range 1 microM-10 microM, SCA40 also caused an increase in the force of atrial contraction. 6. Intracellular electrophysiological recording from guinea-pig isolated trachealis showed that the relaxant effects of SCA40 (1 micro M) were often accompanied by the suppression of spontaneous electrical slow waves but no change in resting membrane potential. When the concentration of SCA40 was raised to 10 micro M, its relaxant activity was accompanied both by slow wave suppression and by plasmalemmal hyperpolarization.7. SCA40 (10 nM- 100 micro M) more potently inhibited the activity of cyclic AMP phosphodiesterase (PDE)than that of cyclic GMP PDE derived from homogenates of guinea-pig trachealis. Theophylline(1 micro M- 1O mM) also inhibited these enzymes but was less potent than SCA40 in each case and did not exhibit selectivity for inhibition of cyclic AMP hydrolysis.8. Tested against the activity of the isoenzymes of cyclic nucleotide PDE derived from human blood cells and lung tissue, SCA40 proved highly potent against the type III isoenzyme. It was markedly less potent against the type IV and type V isoenzymes and even less potent against the isoenzymes types I and II.9. It is concluded that the tracheal relaxant action of SCA40 (1 nM- 1 micro M) does not involve the activation of beta-adrenoceptors or P1 or P2 purinoceptors. Furthermore, this action is unlikely to depend upon the opening of BKca channels with consequent cellular hyperpolarization and voltage-dependent inhibition of Ca2+ influx. The tracheal relaxant action of SCA40 (up to 1 micro M) is more likely to depend upon its selective inhibition of the type III isoenzyme of cyclic nucleotide PDE. At concentrations above 1 micro M, SCA40 exerts more general inhibition of the isoenzymes of cyclic nucleotide PDE and may then promote the opening of BKca channels.

Regulation of cyclic AMP phosphodiesterase activity by particulate protein tyrosine kinase and phosphotyrosine phosphatase activities sensitive to sodium orthovanadate.

Sodium orthovanadate (vanadate) stimulated cAMP phosphodiesterase (PDE) and protein tyrosine kinase (PTK) activities and inhibited the phosphotyrosine phosphatase (PTPase) activity in the particulate of isolated rat fat pads. Okadaic acid never showed any increase in the PDE activity up to 1 microM. Amiloride inhibited in part both stimulations of PDE and PTK activities by vanadate. The particulate PTK activity had an optimal divalent ion requirement of 15 mM Mg+2+2 mM Mn+2 in the assay medium and was not inhibited by 1 mM N-ethylmaleimide, suggesting it to be a different type from the insulin receptor and cytosolic PTK activities. The PDE, PTK, and PTPase active fractions were separated from the solubilized particulate fraction on a DEAE-Sephacel column. PDE activity was increased by the addition of the PTK active fraction. A further increase was observed by using the PTK active fraction pretreated with 1 mM vanadate. In contrast, the addition of PTPase active fraction decreased the PDE activity. This decrease disappeared by using the PTPase active fraction pretreated with 1 mM vanadate. These results suggest that the PDE activity is in part regulated through a process involving the particulate PTK and PTPase activities sensitive to vanadate.

Changes of calmodulin concentration and cyclic 3′, 5′-nucleotide phosphodiesterase activities in cardiac muscle of hyper- and hypothyroid rats

To investigate the effect of thyroid hormone on cardiac muscle dysfunction in hyper- and hypothyroid states, we evaluated cyclic 3',5'-nucleotide metabolism by measuring cyclic 3',5'-nucleotide phosphodiesterase activity and calmodulin concentrations in the cardiac muscles of hyper- and hypothyroid rats. Cyclic AMP (cAMP) concentration was significantly high in the cardiac muscle of hyperthyroid rats and low in that from hypothyroid rats compared with control rats. Cyclic AMP and cyclic GMP phosphodiesterase activities were significantly decreased in the soluble fraction of cardiac muscle from hyperthyroid rats and markedly increased in this fraction in hypothyroid rats compared with normal animals. Calmodulin concentration was high in hyperthyroid and low in hypothyroid rats. It was concluded from these findings that low cAMP-phosphodiesterase activity might, in part, bring about the high concentration of cAMP. Calmodulin was significantly high in the cardiac muscle of hyperthyroid rats and the reverse was the case in hypothyroid rats compared with normal rats. The implication is that, in hyper- and hypothyroid states, these changes may play an important role in cardiac function via their effect on cyclic nucleotide and Ca2+ metabolism.

Identification of two splice variant forms of type-IVB cyclic AMP phosphodiesterase, DPD (rPDE-IVB1) and PDE-4 (rPDE-IVB2) in brain: selective localization in membrane and cytosolic compartments and differential expression in various brain regions.

In order to detect the two splice variant forms of type-IVB cyclic AMP phosphodiesterase (PDE) activity, DPD (type-IVB1) and PDE-4 (type-IVB2), anti-peptide antisera were generated. One set ('DPD/PDE-4-common'), generated against a peptide sequence found at the common C-terminus of these two PDEs, detected both PDEs. A second set was PDE-4 specific, being directed against a peptide sequence found within the unique N-terminal region of PDE-4. In brain, DPD was found exclusively in the cytosol and PDE-4 exclusively associated with membranes. Both brain DPD and PDE-4 activities, isolated by immunoprecipitation, were cyclic AMP-specific (KmcyclicAMP: approximately 5 microM for DPD; approximately 4 microM for PDE-4) and were inhibited by low rolipram concentrations (K1rolipram approximately 1 microM for both). Transient expression of DPD in COS-1 cells allowed identification of an approx. 64 kDa species which co-migrated on SDS/PAGE with the immunoreactive species identified in both brain cytosol and membrane fractions using the DPD/PDE-4-common antisera. The subunit size observed for PDE-4 (approx. 64 kDa) in brain membranes was similar to that predicted from the cDNA sequence, but that observed for DPD was approx. 4 kDa greater. Type-IV, rolipram-inhibited PDE activity was found in all brain regions except the pituitary, where it formed between 30 and 70% of the PDE activity in membrane and cytosolic fractions when assayed with 1 microM cyclic AMP, PDE-4 formed 40-50% of the membrane type-IV activity in all brain regions save the midbrain (approx. 20%). DPD distribution was highly restricted to certain regions, providing approx. 35% of the type-IV cytosolic activity in hippocampus and 13-21% in cortex, hypothalamus and striatum with no presence in brain stem, cerebellum, midbrain and pituitary. The combined type-IVB PDE activities of DPD and PDE-4 contributed approx. 10% of the total PDE activity in most brain regions except for the pituitary (zero) and the mid-brain (approx. 3%. The isolated cDNAs for DPD and PDE-4 appear to reflect transcription products which are expressed in vivo in brain. The unique N-terminal domain of PDE-4 is suggested to target this PDE to membranes in brain. Type-IVB PDEs are differentially expressed in various brain regions, indicating that there are tissue-specific controls on both the expression of the gene and the splicing of its products.

Milrinone, a cyclic AMP-phosphodiesterase inhibitor, has differential effects on regional myocardial work and oxygen consumption in experimental left ventricular hypertrophy.

The aim was to test the hypothesis that local myocardial work and O2 consumption would respond differentially to milrinone, a selective cyclic AMP-phosphodiesterase inhibitor, in left ventricular hypertrophy due to differences in myocardial cyclic AMP-phosphodiesterase activity. The effect of milrinone on regional segment work and regional O2 consumption was measured in 12 open chest anaesthetised dogs with left ventricular hypertrophy induced by valvular aortic stenosis and in 10 age matched control dogs. Regional myocardial work was calculated as the integrated product of instantaneous force development (miniature transducer) and segment shortening (sonomicrometer). Regional O2 consumption was calculated from coronary blood flow (radiolabelled microspheres) and O2 saturations in small regional vessels (microspectrophotometry). Low Km phosphodiesterase activity was assayed by measuring the hydrolysis of radiolabelled cyclic AMP. Milrinone increased left ventricular dP/dtmax by approximately 60-70% in both control [2808(SEM 314) to 4584(660) mm Hg.s-1] and left ventricular hypertrophy [3279(258) to 5589(470) mm-Hg.s-1]. Regional work increased significantly in control [612(88) to 955(101) g.mm.min-1], while the increase was not significant in left ventricular hypertrophy [859(139) to 974(172) g.mm.min-1]. Regional O2 consumption increased significantly with milrinone in left ventricular hypertrophy [8.1(1.2) to 13.1(2.4) ml O2.min-1.100 g-1], but the increase was not significant in control [6.9(1.2) to 7.4(1.0) ml O2.min-1.100 g-1]. Myocardial stiffness during ejection was increased by milrinone to a significantly greater extent in animals with left ventricular hypertrophy. These effects were not related to differences in cyclic AMP-phosphodiesterase activity between control hearts and hearts with left ventricular hypertrophy [393(45) v 402(36) pmol.mg protein-1.1]. Differences between the hypertrophied and normal canine myocardium in response to milrinone are either due to altered levels of cyclic AMP production in left ventricular hypertrophy, to effects of milrinone that are unrelated to cyclic AMP-phosphodiesterase inhibition, or to other differences in hypertrophied hearts. The greater stiffness of the myocardium in left ventricular hypertrophy may require a greater energy expenditure to increase the amount of work it performs.

The identification of apparently novel cyclic AMP and cyclic GMP phosphodiesterase activities in guinea‐pig tracheal smooth muscle

Phosphodiesterase (PDE) activities that were capable of hydrolysing cyclic AMP (Km = 6.8 +/- 2 microM) and cyclic GMP (Km = 6.7 +/- 1.6 microM) were isolated from tracheal smooth muscle. These enzyme(s) activities were insensitive to stimulation by calcium/calmodulin and to inhibition by cyclic GMP, rolipram (type IV inhibitor) and siguazodan (type III inhibitor). Zaprinast was a relatively poor inhibitor of both cyclic AMP and cyclic GMP hydrolysis (IC50 = 46 +/- 9 microM and 45 +/- 14 microM respectively). These results suggest that tracheal smooth muscle may contain an apparently novel PDE. However, KCl (30 mM) which facilitates calcium entry in cells, depressed bradykinin-stimulated intracellular cyclic AMP formation, suggesting that the type I PDE may be functionally present. We suggest that considerable caution be exercised in identifying apparently novel PDE isoforms.

Evidence that cyclic AMP is involved in the hypersensitive response of Medicago sativa to a fungal elicitor

In response to challenge with a glycoprotein elicitor from the phytopathogenic fungus, Verticillium alboatrum, synthesis of the phytoalexin medicarpin is induced in Medicago sativa. Treatment of seedlings with the cell-permeating cyclic AMP analogue, dibutyryl cyclic AMP, resulted in the stimulation of phenylalanine ammonia lyase activity and induction of medicarpin synthesis. Challenge of M. sativa cell suspension cultures with fungal elicitor resulted in a significant, transient increase in intracellular cyclic AMP content, together with a pulse of adenylyl cyclase activity, both within a few minutes of the elicitor challenge, and a subsequent increase in cyclic AMP phosphodiesterase activity. Incubation of a membrane fraction from the M. sativa cells with the fungal elicitor resulted in a dose-dependent stimulation of the adenylyl cyclase activity. These observations are discussed in the context of the signal transduction mechanism with the M. sativa cellular defence system.

Bradykinin-dependent activation of adenylate cyclase activity and cyclic AMP accumulation in tracheal smooth muscle occurs via protein kinase C-dependent and -independent pathways.

Treatment of cultured tracheal smooth-muscle cells (TSM) with phorbol 12-myristate 13-acetate (PMA) (100 nM) or bradykinin (100 nM) elicited enhanced basal and guanosine 5'-[beta gamma-imido]-triphosphate-stimulated adenylate cyclase activities in subsequently isolated membranes. Combined stimulation of cells was non-additive, indicating that both agents activate adenylate cyclase via similar routes. Both PMA (100 nM) and bradykinin (100 nM) allowed the alpha subunit of Gs to act as a more favourable substrate for its cholera-toxin-catalysed ADP-ribosylation in vitro. PMA was without effect on intracellular cyclic AMP in control cells. However, constitutive activation of Gs by treatment in vivo with cholera toxin (0.5 ng/ml, 18 h) sensitized the cells to PMA stimulation, resulting in a concentration-dependent increase in intracellular cyclic AMP accumulation (EC50 = 7.3 +/- 2.5 nM, n = 5). Bradykinin also elicited a concentration-dependent increase in intracellular cyclic AMP (EC50 = 63.3 +/- 14.5 nM, n = 3). Constitutive activation of Gs resulted in an increased maximal response (10-fold) and potency (EC50 = 6.17 +/- 1.6 nM, n = 3) to bradykinin. This response was not affected by the B2-receptor antagonist, NPC567 [which selectively blocks bradykinin-stimulated phospholipase C (PLC), with minor activity against phospholipase D (PLD) activity]. Des-Arg9-bradykinin (a B1-receptor agonist) was without activity. These results suggest that the receptor sub-type capable of activating PLD may also be stimulatory for cyclic AMP accumulation. Furthermore, pre-treatment of the cells with butan-l-ol (0.3%, v/v), which traps phosphatidate derived from PLD reactions, blocked the bradykinin-stimulated increase in intracellular cyclic AMP. These studies suggest that there may be a causal link between PLD-derived phosphatidate and the positive modulation of adenylate cyclase activity. In support of this, the concentration-dependence for bradykinin-stimulated adenylate cyclase activity was identical with that of bradykinin-stimulated phospholipase D activity (EC50 = 5 nM). Bradykinin, but not PMA, was also capable of eliciting the inhibition of cyclic AMP phosphodiesterase activity in TSM cells (EC50 > 100 nM) via an unidentified mechanism. These studies indicate that cross-regulation between the cyclic AMP pathway and phospholipid-derived second messengers in TSM cells does not occur as a consequence of PLC-catalysed PtdIns(4,5)P2 hydrolysis, but may involve, in part, PLD-catalysed phosphatidylcholine hydrolysis.

The Effects of Theophylline on Aequorin Light Transients and Force in the Isolated Dog Right Ventricular Myocardium

Experiments were carried our to investigate the changes in intracellular Ca 2+ transients associated with biphasic contractions that were elicited during interaction of theophylline with isoproterenol in the dog ventricular myocardium. For this purpose, effects of theophylline and isoproterenol on aequorin light transients and isometric contractions were assessed in the isolated canine ventricular trabeculae, superficial cells of which had been microinjected with the Ca 2+ sensitive bioluminescent protein aequorin. The positive inotropic effect of theophylline (0.1-0.3 mM) was consistently associated with an increase in the amplitude of aequorin light transients. Theophylline at concentrations of 0.6 mM and higher decreased the amplitude of aequorin light transients, but the force of contraction increased further in association with a prominent prolongation of time to peak force. Theophylline (0.3 mM) enhanced the forskolin-induced increase in aequorin light transients and force. Theophylline (2 mM) inhibited the isoproterenol-induced increase in aequorin light transients associated with early phase of contraction in a reversible manner. A late phase of aequorin light transients was induced in association with late phase of contraction in the presence of both isoproterenol and theophylline. Thus, both the early and late phase of contraction were accompanied by corresponding phases of aequirin light transients. The relation between the amplitude of force and Ca 2+ transients was markedly different and the late phase of contraction was associated with much lower aequorin light transients. The late phase of aequorin light transients induced by theophylline at a high concentration (10 mM) was enhance by isoproterenol. These results indicate that theophylline (0.1-0.3 mM) increases the amplitude of Ca 2+ transients through an accumulation of cyclic AMP by inhibition of the cyclic AMP phosphodiesterase activity. In concentrations of 0.6 mM and higher theophylline decreases the amplitude of the early phase aequorin light transients probably by inhibition of release of Ca 2+ from the sarcoplasmic reticulum and induces simultaneously the late phase of contraction that may be associated with an increase in responsiveness to Ca 2+ of myofibrils.

Growth of S49 wild type cells in 3 nM epinephrine increases cyclic AMP phosphodiesterase activity

In this communication we report that intact cell measurements of cAMP decay have shown an increase averaging over 80% in the cAMP decay constant (k dy) in intact S49 WT cells following a 24 hour growth in 3 nM epinephrine (24 hr/3 nM epinephrine). A comparable percentage increase was seen in cell free PDE activities from cells similarly treated. The enhanced PDE activity and k dy acre detectable after 6 hours of incubation with 3 nM epinephrine, and were maximal by 24 hours of incubation. Lysate PDE activity returned to control levels within 24 hours after the cells were transferred to epinephrine-free growth medium. The increased PDE activity present in S49 WT cells after 24 hr/3 nM epinephrine was the result of increased concentrations of the enzymes already present or the expression of very similar enzymes, rather than the expression of enzymes with markedly different characteristics. Firstly, the apparent K m values for the PDE's in lysates from control and 24 hr/3 nM epinephrine cells were both in the region of 1 μM. Secondly, PDE activities in lysates from 24 hr/control and 24 hr/3 nM epinephrine S49 WT cells showed similar sensitivities to PDE inhibitors. There was support for the hypothesis that the increase in PDE activity in 24 hr/3 nM epinephrine S49 WT cells is dependent on the presence of cAPK. That is, although 24 hr/3nM epinephrine caused a distinct homologous desensitization of adenylate cyclase in S49 kin cells, neither k dy nor lysate PDE activity were affected. Additionally, 24 hours incubation of S49 WT cells with 3 μM dibutyryl cAMP resulted in increased PDE activity. Finally, the stimulatory effect of 24 hr/3 nM epinephrine on PDE activity was inhibited in the presence of cycloheximide, suggesting the involvement of protein synthesis in the process. This study shows that prolonged treatment with very low concentrations of epinephrine results in an increase in PDE activity which had a significant effect on cAMP accumulation. The increases were quantified by intact cell and cell free measurements, with kinetic data which were consistent with the hypothesis that the increased PDE activity in 24 hr/3 nM epinephrine cells reflected an increase PDE synthesis which was dependent on activation of the cAPK.