Cyclic GMP Phosphodiesterase Activity Research Articles

Changes in cyclic GMP level and phosphodiesterase activity during follicular development in the rat ovary.

Guanosine 3',5'-cyclic monophosphate (cGMP), as a second messenger, plays potential roles in ovarian functions. To elucidate the role of phosphodiesterase (PDE) in cGMP signaling during ovarian follicular development, the present study was conducted to investigate ovarian cGMP level and cGMP-PDE activity by radioimmunoassay (RIA) in postnatal rats, immature rats during gonadotropin-primed follicular development, ovulation and luteinization, adult rats during normal estrous cycle, and aged rats that spontaneously developed persistent estrus (PE). All four rat models were confirmed by histological examination of one ovary, and the other ovary was used for RIA. In postnatal rats, cGMP level was high at birth and decreased dramatically by Day 5, and then, it increased maximally at Day 10 and declined at Day 21. However, cGMP-PDE activity did not significantly change during Days 1 to 10, but increased significantly on Day 21. In immature female rats, cGMP level markedly decreased upon treatment with equine chorionic gonadotropin (eCG), while cGMP-PDE activity did not show any significant changes; however, ovarian cGMP level and cGMP-PDE activity increased after injection of an ovulatory dose of human chorionic gonadotropin (hCG) for induction of ovulation and luteinization. In adult rats during normal estrous cycle, cGMP level was high on proestrus and metestrus days, while cGMP-PDE activity was high on estrus day. In PE rats, ovarian cGMP level was similar to that in adult rats on estrus and diestrus days but lower than that on proestrus and metestrus days; ovarian cGMP-PDE activity was lower than that on estrus days but similar as the other estrous cycle days. In addition, there was a significant negative correlation between ovarian cGMP level and cGMP-PDE activity during normal estrous cycles in the adult rat (r = -0.7715, N = 16, P < 0.05), but not in the postnatal rat (r = -0.1055, N = 20, P > 0.05). Together, the results of our present study indicated that ovarian cGMP levels were not dependent on cGMP-PDE activity during early postnatal development, but highly dependent on cGMP-PDE activity in the adult rat. This implies that mechanisms of cGMP signaling involved in ovarian functions are stage-specific in the rat.

Tokay Gecko Photoreceptors Achieve Rod-Like Physiology with Cone-Like Proteins†

The retinal photoreceptors of the nocturnal Tokay gecko (Gekko gekko) consist exclusively of rods by the criteria of morphology and key features of their light responses. Unlike cones, they display robust photoresponses and have relatively slow recovery times. Nonetheless, the major and minor visual pigments identified in gecko rods are of the cone type by sequence and spectroscopic behavior. In the ongoing search for the molecular bases for the physiological differences between cones and rods, we have characterized the molecular biology and biochemistry of the gecko rod phototransduction cascade. We have cloned cDNAs encoding all or part of major protein components of the phototransduction cascade by RT-PCR with degenerate oligonucleotides designed to amplify cone- or rod-like sequences. For all proteins examined we obtained only cone-like and never rod-like sequences. The proteins identified include transducin alpha (Galphat), phosphodiesterase (PDE6) catalytic and inhibitory subunits, cyclic nucleotide-gated channel (CNGalpha) and arrestin. We also cloned cDNA encoding gecko RGS9-1 (Regulator of G Protein Signaling 9, splice variant 1), which is expressed in both rods and cones of all species studied but is typically found at 10-fold higher concentrations in cones, and found that gecko rods contain slightly lower RGS9-1 levels than mammalian rods. Furthermore, we found that the levels of GTPase accelerating protein (GAP) activity and cyclic GMP (cGMP) phosphodiesterase activity were similar in gecko and mammalian rods. These results place substantial constraints on the critical changes needed to convert a cone into a rod in the course of evolution: The many features of phototransduction molecules conserved between those expressed in gecko rods and those expressed in cones cannot explain the physiological differences, whereas the higher levels of RGS9-1 and GAP activity in cones are likely among the essential requirements for the rapid photoresponses of cones.

HIV-1 coat protein gp120 decreases NO-dependent cyclic GMP accumulation in rat brain astroglia by increasing cyclic GMP phosphodiesterase activity

The human immunodeficiency virus type-1 (HIV-1) coat glycoprotein gp120 has been proposed as a likely etiologic agent of HIV-associated dementia (HAD). The pathogenic mechanisms underlying HAD have not yet been fully elucidated, but different evidences indicate that glial cells play an essential role in the development and amplification of the disease. The NO/cyclic GMP (cGMP) system is a widespread signal transduction pathway in the CNS involved in numerous physiological and pathological functions. Increased expression of NO synthase has been reported in the brain of AIDS patients and in cultured rodent glial cells exposed to gp120. The aim of this study was to investigate if gp120 could cause alterations in the metabolism of the NO physiological messenger cGMP that could contribute to the pathogenesis of HAD. Here, we show that long-term treatment (more than 24 h) of rat cerebellar astrocyte-enriched cultures with gp120 (10 nM) induces changes in the cultured cells—astrocyte stellation and proliferation of ameboid microglia—compatible with the acquisition of a reactive phenotype and reduces the capacity of the astrocytes to accumulate cGMP in response to NO in a time-dependent manner (maximal after 72 h). Measurements in cell extracts show that gp120 enhances Ca 2+-independent cGMP phosphodiesterase activity by 80–100% without significantly affecting soluble guanylyl cyclase (sGC). Experiments in whole cells using specific phosphodiesterase inhibitors indicate that the viral protein increases the activity of cGMP specific phosphodiesterase 5.

Antiplatelet effects of KW-7, a new inhibitor of cyclic nucleotide phosphodiesterases

The antiplatelet effect of a new synthetic compound, 8,9-dimethoxyl-1-(4-methoxy-phenyl)-5,6-dihydro-pyrrolo[2,1- a]isoquinoline-2,3-dione (KW-7), was determined in rabbit platelets. KW-7 concentration-dependently prevented platelet aggregation caused by arachidonic acid, collagen, platelet-activating factor, and thrombin. KW-7 induced a substantial increase in cyclic AMP levels and a smaller increase in cyclic GMP levels in platelets. In platelet homogenates, KW-7 inhibited both cyclic AMP- and cyclic GMP-phosphodiesterase activities. The antiplatelet effect of KW-7 was reversed by SQ22536 (an inhibitor of adenylate cyclase) and H89 (an inhibitor of protein kinase A) but not by ODQ (an inhibitor of soluble guanylate cyclase). These data suggest that the antiplatelet effect of KW-7 is cyclic AMP-dependent, and is through inhibition of platelet phosphodiesterases. In addition, KW-7 inhibited arachidonic acid-stimulated thromboxane production; this effect was associated with an increase in prostaglandin D 2 levels indicating KW-7 is also an inhibitor of thromboxane synthase. The dual inhibition of KW-7 on phosphodiesterase and thromboxane synthase might provide an attractive target in developing antiplatelet drugs.

Reciprocal regulation of cyclic GMP content by cyclic GMP-phosphodiesterase and guanylate cyclase in SHR with CsA-induced nephrotoxicity.

1. The effect of the immunosuppressant drug, cyclosporin A (CsA), on the nitric oxide (NO)-cyclic GMP pathway was examined in spontaneous hypertensive rats (SHR). 2. CsA (50 mg kg(-1)) treatment for 14 days induced typical CsA nephrotoxicity, which was characterized by morphological changes in the glomerulus and proximal tubule as well as an abnormality of creatinine clearance, FENa and BUN. 3. CsA significantly decreased both NOS activity in the kidney and NOx contents in urine, but significantly increased cyclic GMP content in the kidney. 4. A marked change in two kinds of enzyme, which contribute towards the increase in cyclic GMP in tissue, namely, a decrease in cyclic GMP-phosphodiesterase activity and increase in guanylate cyclase activity, was observed in the kidney treated with CsA. 5. In the isolated perfused kidney, a decreased in perfusion pressure induced by SNP in the kidney isolated from CsA group was significantly greater than that of control. 6. There seem to exist a reciprocal mechanism to maintain cyclic GMP content via both a decrease in cyclic GMP degradation and an increase in synthesis of cyclic GMP in the kidney treated with CsA. This mechanism is likely to be playing an important role to regulate the homeostasis in the kidney with CsA nephrotoxicity.

Effects of the soluble guanylyl cyclase activator, YC-1, on vascular tone, cyclic GMP levels and phosphodiesterase activity.

The vasomotor and cyclic GMP-elevating activity of YC-1, a novel NO-independent activator of soluble guanylyl cyclase (sGC), was studied in isolated rabbit aortic rings and compared to that of the NO donor compounds sodium nitroprusside (SNP) and NOC 18. Similarly to SNP and NOC 18, YC-1 (0.3-300 microM) caused a concentration-dependent, endothelium-independent relaxation that was greatly reduced by the sGC inhibitor 1-H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ 10 microM; 59% inhibition of dilation induced by 100 microM YC-1) suggesting the activation of sGC as one mechanism of action. Preincubation with YC-1 (3 and 30 microM) significantly increased the maximal dilator responses mediated by endogenous NO in aortic rings that was released upon exposure to acetylcholine, and enhanced the dilator response to the exogenous NO-donors, SNP and NOC 18, by almost two orders of magnitude. Vasoactivity induced by SNP and YC-1 displayed different kinetics as evidenced by a longlasting inhibition by YC-1 (300 microM) on the phenylephrine (PE)-induced contractile response, which was not fully reversible even after extensive washout (150 min) of YC-1, and was accompanied by a long-lasting elevation of intracellular cyclic GMP content. In contrast, SNP (30 microM) had no effect on the vasoconstrictor potency of PE, and increases in intravascular cyclic GMP levels were readily reversed after washout of this NO donor compound. Surprisingly, YC-1 not only activated sGC, but also affected cyclic GMP metabolism, as it inhibited both cyclic GMP break down in aortic extracts and the activity of phosphodiesterase isoforms 1-5 in vitro. In conclusion, YC-1 caused persistent elevation of intravascular cyclic GMP levels in vivo by activating sGC and inhibiting cyclic GMP break down. Thus, YC-1 is a highly effective vasodilator compound with a prolonged duration of action, and mechanisms that are unprecedented for any previously known sGC activator.

Cyclic AMP and cyclic GMP phosphodiesterase inhibition by an antiplatelet agent, 6-[(3-methylene-2- oxo-5-phenyl-5-tetrahydrofuranyl)methoxy]quinolinone (CCT-62)

The antiplatelet activity of (6-[(3-methylene-2- oxo-5-phenyl-5-tetrahydrofuranyl) methoxy]quinolinone) (CCT-62) was determined in vitro in rabbit platelets. CCT-62 inhibited rabbit platelet aggregation and ATP release caused by thrombin (0.1 U/ml), platelet-activating factor (2 ng/ml), collagen (10 μg/ml), arachidonic acid (100 μM), and 9,11-dideoxy-9 α,11 α-methanoepoxy prostaglandin F 2 α (1 μM) in a concentration-dependent manner. The IC 50 values for platelet aggregation were 18.4±4.5, 10.1±1.6, 3.0±0.9, 1.5±0.3 and 1.0±0.3 μM, respectively. In addition, CCT-62 disaggregated the clumped platelets caused by these aggregation inducers. It also inhibited phosphoinositide breakdown and intracellular calcium elevation induced by the above platelet aggregation inducers. CCT-62 increased intracellular cyclic AMP and cyclic GMP levels in a concentration- and time-dependent manner. Furthermore, it potentiated cyclic AMP formation caused by prostaglandin E 1 but not that caused by 3-isobutyl-1-methylxanthine. CCT-62 did not affect adenylate or guanylate cyclase but inhibited cyclic AMP- and cyclic GMP-phosphodiesterase activities. The antiplatelet effect of CCT-62 was reversed by a protein kinase A inhibitor, N-[2-( P-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89). This data clearly indicated that CCT-62 is an inhibitor of phosphodiesterases and that its antiplatelet effect is mainly mediated by elevation of cyclic AMP levels.

Pharmacological activity of PF-904 in guinea-pig in vivo, and on human bronchus and neutrophils in vitro

The effects of PF-904 (4-amino-1-ethyl-6-methylpyrazino[2,3-c][1,2,6]thiadiazine 2,2-dioxide), a pyrazinothiadiazine derivative, were examined in guinea-pig airways in vivo, in human isolated bronchus and human polymorphonuclear leukocytes. PF-904 (12.5–200 mg kg −1, intraduodenal) reduced bronchoconstriction in response to histamine, arachidonic acid, platelet-activating factor (PAF) and methacholine. PF-904 (50–200 mg kg −1) prevented PAF-induced airways hyperreactivity and inhibited antigen-induced bronchoconstriction, airway microvascular leakage and eosinophil lung accumulation, but antigen-induced airways hyperresponsiveness was not reduced. PF-904 (1 μM–1 mM) produced complete inhibition of spontaneous (−log EC 50=3.57±0.04; n=10) and histamine-stimulated tone (−log EC 50=3.66±0.07; n=10) of human isolated bronchus. Glibenclamide (10 μM) or precontraction with KCl (80 mM) did not impede PF-904-induced bronchial relaxation. PF-904 inhibited cyclic AMP (−log IC 50=2.83±0.25; n=8) and cyclic GMP (−log IC 50=2.90±0.21; n=8) phosphodiesterase activity in human bronchus. The activity of type IV phosphodiesterase was inhibited by PF-904 (−log IC 50=3.43±0.11; n=3). PF-904 also inhibited superoxide release by N-formylmethionyl–leucyl–phenylalanine-stimulated human polymorphonuclear leukocytes, but the maximal effect was ~50% of that produced by rolipram (10 μM). This profile of activities of PF-904 suggests that this compound has potential therapeutic value as an anti-asthma drug.

Ca 2+/calmodulin-dependent cyclic GMP phosphodiesterase activity in granule neurons and astrocytes from rat cerebellum

Cyclic GMP (cGMP) formation induced by agonist stimulation of Ca 2+/calmodulin-dependent nitric oxide (NO) synthase type I is known to occur in both granule cell and astrocyte cultures from rat cerebellum. Here we show that in these same cells cGMP is predominantly hydrolyzed by a Ca 2+/calmodulin-dependent phosphodiesterase. At 10 μM cGMP, Ca 2+ (25 μM) stimulated basal (Ca 2+-independent) phosphodiesterase activity about 6 times in granular neurons and 15 times in astrocytes. The calmodulin antagonist calmidazolium blocked the Ca 2+-dependent phosphodiesterase activity and exogenous calmodulin increased 3–4-fold the stimulatory potency of Ca 2+ in both cell types (EC 50 values 1.26±0.20 and 1.50±0.42 μM in the absence and 0.38±0.11 and 0.39±0.14 μM in the presence of 1 μM calmodulin, for neurons and astrocytes, respectively). In both cell types K m values for cGMP at 25 μM Ca 2+ were similar (1.72±0.20 and 1.92±0.09 μM) and phosphodiesterase activities were inhibited by isozyme-selective phosphodiesterase inhibitors with potencies analogous to those described for Ca 2+/calmodulin-phosphodiesterases or phosphodiesterase type 1 isoforms in other preparations. The nonselective phosphodiesterase inhibitor 3-isobutyl-1-methylxantine (IBMX) effectively blocked the Ca 2+/calmodulin-phosphodiesterase activity in granule cell and astrocyte extracts (IC 50 values at 1 μM cGMP: 31±10 μM and 46±6 μM, respectively), in contrast to the apparent inability of this compound to inhibit the Ca 2+-dependent activity reported in whole brain extracts. These results demonstrate that comparable phosphodiesterase type 1 activities are found in the cytosols of cerebellar granule cells and astrocytes and suggest that these activities may play an important role in controlling cGMP levels in cells where the Ca 2+-dependent NO synthase type I is stimulated. © 1997 Elsevier Science B.V. All rights reserved.

Cyclic GMP accumulation in pulmonary microvascular endothelial cells measured by intact cell prelabeling.

Cyclic GMP accumulation in cultured rat pulmonary microvascular endothelial cells (RPMVEC) was studied with a new prelabeling method developed using intact platelets and smooth muscle cells (1). [3H]-hypoxanthine was used to radiolabel the cellular guanine nucleotide pool. Neutral alumina and Dowex-50 double column chromatography was used to purify and quantitate the levels of [3H]-cyclic GMP. Changes in cyclic GMP metabolism in short and long term RPMVEC cultures were studied using rat atrial naturetic factor 8-33 (ANF) and sodium nitroprusside (SNP) in the presence and absence of cyclic nucleotide (CN) phosphodiesterase (PDE) inhibitors. In RPMVEC exogenous hypoxanthine was incorporated into both low (65% uptake) and high (34% uptake) passage cells in a time-dependent manner reaching maximum incorporation near 8 hours. Basal cyclic GMP values in both groups were 0.003% of the total cellular tritium (9 x 10(6) and 4 x 10(6) cpm/10(6) cells, respectively). ANF treatment of prelabeled RPMVEC resulted in a 10- to 12-fold increase in [3H]-cyclic GMP in the absence of CN PDE inhibitors (EC50 = 5.4 nM). However, incubation with SNP showed no changes in cellular cyclic GMP accumulation. Several relatively selective CN PDE inhibitors had no effect on ANF or SNP induced cyclic GMP accumulation in RPMVEC. The ANF induced cGMP accumulation was verified by radioimmunoassay. These studies confirm the utility of the hypoxanthine prelabeling technique to monitor intact microvascular EC cyclic GMP accumulation. Cultured RPMVEC show little or no functional soluble guanylate cyclase or cyclic GMP PDE activity.

Increased guanylate cyclase activity is associated with an increase in cyclic guanosine 3',5'-monophosphate in left ventricular hypertrophy.

Left ventricular hypertrophy (LVH) produced by aortic valve plication leads to increased myocardial cyclic GMP. We tested whether this was a result of increased soluble guanylate cyclase activity or nitric oxide (NO) synthase and its functional consequences. We used the nitric oxide donor 3-morpholino-sydnonimine (SIN-1) or the NO synthase inhibitor NG-nitro-l-arginine methyl ester (L-NAME) in 12 control and 12 LVH anesthetized open-chest mongrel dogs. L-NAME (6 mg/kg) or SIN-1 (1 microgram/kg per min) was infused into the left anterior descending coronary artery and regional segment work and cyclic GMP levels were determined. In vitro myocardial guanylate cyclase sensitivity (0.43 +/- 0.04 to 0.28 +/- 0.04 mM [EC50]) and maximal activity (10.1 +/- 2.9 to 25.5 +/- 6.5 pmol/mg protein per min) were significantly increased in LVH as compared with control animals in response to nitroprusside stimulation, but cyclic GMP-phosphodiesterase activity was similar. In LVH dogs, basal cyclic GMP was significantly elevated in vivo when compared with controls. Treatment of dogs with SIN-1 resulted in a significant increase in cyclic GMP in control (1.09 +/- 0.12 to 1.48 +/- 0.19 pmol/gram) and a greater increase in the LVH group (1.78 +/- 0.16 to 3.58 +/- 0.71 pmol/g). L-NAME had no effect on myocardial cyclic GMP levels in control or LVH dogs. Segment work decreased in the control group after SIN-1 (1,573 +/- 290 to 855 +/- 211 grams x mm/min). LVH dogs showed no decrement in work as a result of treatment with SIN-1. L-NAME did not cause significant changes in myocardial cyclic GMP, O2 consumption, or work in either control or LVH dogs, but vascular effects were evident. SIN-1 increased cyclic GMP, and with greater effect on LVH; however, this resulted in a decrement in function only in the control group. The greater increased cyclic GMP in LVH dogs is not related to increased NO production, but is related to significantly higher sensitivity and maximal activity of soluble myocardial guanylate cyclase.

Melatonin effect on the cyclic GMP system in the golden hamster retina

Melatonin effect on retinal cyclic GMP accumulation, guanylate cyclase activity, cyclic GMP content and cyclic GMP phosphodiesterase activity was examined in the Syrian hamster retina. Melatonin increased significantly cyclic GMP accumulation at picomolar concentrations and in a time-dependent manner. The kinetic analysis of guanylate cyclase activity revealed a significant increase of both apparent V max and K m, induced by 10 nM melatonin. The effect of melatonin was higher in the absence, than in the presence of the phoshodiesterase inhibitor (IBMX), suggesting an effect on cyclic GMP catabolism. Phosphodiesterase activity was significantly decreased by melatonin. The results show a dual effect of melatonin on cyclic GMP levels, i.e. by increasing the synthesis and inhibiting the degradation, both resulting in an increase of cyclic GMP levels. Taking into account the key role of cyclic GMP in visual mechanisms, the results would suggest the participation of melatonin in retinal physiology.

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.

Effects of topical methylene blue on cyclic GMP level, blood flow, and O2 consumption in focal cerebral ischaemia.

We hypothesized that a decrease in cyclic GMP, a second messenger in the glutamate-nitric oxide pathway, would reduce oxygen consumption and improve O2 balance in the ischaemic cerebral cortex. To test this hypothesis, a study was performed in unilateral middle cerebral artery occluded rats which were assigned to either a control or methylene blue (10(-3) M) group. Regional cerebral blood flow was determined using 14C-iodoantipyrine and regional arterial and venous O2 saturations were determined by microspectrophotometry (n = 6). Cyclic GMP level was measured by radioimmunoassay (n = 8). Guanylate cyclase and cyclic GMP-phosphodiesterase activities were determined in an additional set of control rats (n = 10). The cyclic GMP levels were not different between the ischaemic and contralateral areas in the control group. Compared to the cyclic GMP in the control ischaemic cortex, topical methylene blue significantly decreased the cyclic GMP level by 56% in the ischaemic cortex of the methylene blue group. Ischaemia did not alter the activities of guanylate cyclase but mildly decreased cyclic GMP-phosphodiesterase. The regional cerebral blood flow and O2 consumption in the control group were 50% and 32% lower than those in corresponding contralateral cortex. Topical methylene blue did not alter regional cerebral blood flow and O2 consumption in the ischaemic cortex. Our data showed that cyclic GMP is not a major controller on O2 supply or O2 consumption in the ischaemic brain.

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.

Effects of VA-045, a novel apovincaminic acid derivative, on isolated blood vessels: Cerebroarterial selectivity

We investigated the effects of VA-045, an apovincaminic acid derivative, on isolated blood vessels. VA-045 (10 −7−10 −5M) and vinpocetine (10 −7−10 −5M) inhibited the 64 mM KCl-induced and 10 −6M norepinephrine (NE)-induced contraction of rat aortic strips. VA-045 (10 −7−10 −4M) and vinpocetine (10 −7−10 −4M) inhibited the activity of cyclic AMP and cyclic GMP phosphodiesterase in porcine coronary artery. VA-045 (3×10 −9−3×10 −6M) relaxed the 64 mM KCl-induced contraction of the canine basilar artery without affecting the peripheral arteries. These results indicate that VA-045 selectively dilates canine cerebral artery, and that it may be a useful agent for the treatment of cerebrovascular diseases such as stroke.

Interaction of the catalytic subunit of protein kinase A with the lung type V cyclic GMP phosphodiesterase: Modulation of non-catalytic binding sites

We have previously demonstrated that the catalytic sub-unit of protein kinase A can catalyse a potent activation of partially purified Type V cyclic GMP-specific phosphodiesterase activity ( Burns et al., 1992, Biochem. J. 283, 487–491 ). We now demonstrate that this phosphodiesterase most likely has a sub-unit mass of 90kDa, based upon 32P-cyclic GMP photoaffinity labelling, that activation of the phosphodiesterase does not require the prior binding of cyclic GMP to the phosphodiesterase, and that alkaline phosphatase can reverse the protein kinase A-dependent activation of phosphodiesterase activity. Zaprinast is a mixed inhibitor of non-activated cyclic GMP phosphodiesterase activity. However, inhibition of the protein kinase A-activated phosphodiesterase is competitive. These results suggest that protein kinase A can modulate the inhibitory effects of zaprinast via perturbations of a non-catalytic binding site.

Modulation of cyclic nucleotide phosphodiesterase by dietary fats in rat heart.

Feeding oils of different fatty acid composition modifies the fatty acid composition of cardiac membrane phospholipids, thereby inducing changes in cardiac contractility and altering response of adenylate cyclase to catecholamines. In the present study, the effect of such dietary manipulations on cyclic nucleotide phosphodiesterase, which is involved in the control of cyclic nucleotide intracellular levels and in the control of cardiac contractility, was investigated. Rats were fed either a saturated fatty acid-enriched diet (8 weight percent [%] coconut oil + 2% sunflower oil), an n-6 fatty acid-enriched diet (10% sunflower oil) or an n-3 fatty acid-enriched diet (8% fish oil + 2% sunflower oil). The fatty acid composition of cardiac phospholipids, as well as the nonesterified fatty acid content of heart were markedly altered by the diets. The 18:2n-6 and 20:4n-6 content of cardiac phospholipids was markedly (-49%) depressed by fish oil as compared with sunflower oil feeding, but the nonesterified fatty acid level of heart membrane was lowest in coconut oil-fed rats. In addition, fish oil feeding more drastically depressed the n-6/n-3 fatty acid ratio in the nonesterified fatty acid pool than in cardiac phospholipids. Cyclic AMP phosphodiesterase activity was the lowest in both the particulate and soluble fractions of heart from rats fed sunflower oil, whereas cyclic GMP phosphodiesterase activity was not altered by the diets. Cyclic AMP phosphodiesterase activity was decreased by 18 and 12% in heart membranes of the sunflower oil group as compared to that of the coconut oil and fish oil groups, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Early increase in lymphocyte cyclic nucleotide phosphodiesterase activity upon mitogenic activation of human peripheral blood mononuclear cells.

Cyclic nucleotide phosphodiesterase activity of human peripheral blood mononuclear cells was significantly increased following a short (30 min) incubation with the mitogenic lectin Concanavalin A. Con A stimulated phosphodiesterase activity to the same extent whatever the subcellular compartment (homogenate, cytosol or pellet). Further separation of the Con A-activated mononuclear cells into lymphocyte-enriched and monocyte-enriched populations showed that the Con A-induced increase of phosphodiesterase activity exclusively affected the lymphocyte-enriched population. In lymphocytes, cyclic GMP phosphodiesterase activity was more importantly enhanced by Con A (+275%) than cyclic AMP phosphodiesterase activity (+75%). The increase of both activities occurred as early as from 10 min of Con A incubation and proved to be maximal with Con A doses of 2.5 and 5 micrograms per 10(6) cells, lower and higher doses being less effective. Inhibition experiments with reference inhibitors suggested that, among the high affinity phosphodiesterase isoforms, the cyclic GMP-inhibited enzyme might be more selectively enhanced by Con A than the cyclic AMP-specific, Rolipram-sensitive one. The non-mitogenic lectin Helix pomatia hemagglutinin, was not able to enhance cyclic nucleotide phosphodiesterase activity of human mononuclear cells whereas anti-CD3 monoclonal antibody, although being less effective than Con A, exhibited a significant stimulatory effect. Putting together these results suggest that the early increase in phosphodiesterase activity might be a normal step involved in the mitogenic activation of human lymphocyte.

Impaired capacity of lymphocytic proteins to upregulate β-adrenoceptors in A549 human lung adenocarcinoma cells in respiratory and cutaneous atopic disease

The comparative inhibitory potency of papaverine and Ro 20-1724 (4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone) on cyclic AMP-phosphodiesterase (cAMP-PDE) and cyclic GMP-phosphodiesterase (cGMP-PDE) activities and their effect on the levels of cAMP and cGMP were examined in psoriatic epidermis. At concentrations of 5 × 10-4 M, papaverine inhibited the hydrolysis of both cAMP and cGMP by either the low or high Km psoriatic epidermal PDE nearly 100% (p <.0001) while Ro 20-1724 selectively inhibited the hydrolysis of cAMP 94% (p <.0001) but had no significant effect on cGMP hydrolysis. When keratomed psoriatic epidermal slices were incubated in 5 × 10-4 M papaverine or Ro 20-1724 the tissue levels of cAMP were increased 343% or 1395% respectively (p <.001) with no concomitant change in the levels of cGMP. Selectice inhibition of cAMP hydrolysis by Ro 20-1724 and its greater effectiveness in elevating cAMP levels in slices of psoriatic epidermis Is one explanation for its clinical superiority in treating psoriatic lesions.