Monophosphate Phosphodiesterase Research Articles

A Semiautomated Method for the Assay of Cyclic Adenosine 5′-Monophosphate Phosphodiesterase

A Semiautomated Method for the Assay of Cyclic Adenosine 5′-Monophosphate Phosphodiesterase

Uncoupling of GTP Binding from Target Stimulation by a Single Mutation in the Transducin α Subunit

Glutamic acid-203 of the alpha subunit of transducin (alphaT) resides within a domain that undergoes a guanosine triphosphate (GTP)-induced conformational change that is essential for effector recognition. Changing the glutamic acid to an alanine in bovine alpha(T) yielded an alpha subunit (alpha(T)E203A) that was fully dependent on rhodopsin for GTP-guanosine diphosphate (GDP) exchange and showed GTP hydrolytic activity similar to that measured for wild-type alpha(T). However, unlike the wild-type protein, the GDP-bound form of alpha(T)E203A was constitutively active toward the effector of transducin, the cyclic guanosine monophosphate phosphodiesterase. Thus, the alpha(T)E203A mutant represents a short-circuited protein switch that no longer requires GTP for the activation of the effector target phosphodiesterase.

Recovery kinetics of human rod phototransduction inferred from the two-branched alpha-wave saturation function.

Electroretinographic data obtained from human subjects show that bright test flashes of increasing intensity induce progressively longer periods of apparent saturation of the rod-mediated electroretinogram (ERG) alpha wave. A prominent feature of the saturation function [the function that relates the saturation period T with the natural logarithm of flash intensity (ln I(f)] is its two-branched character. At relatively low flash intensities (I(f) below approximately 4 x 10(4) scotopic troland second), T increases approximately in proportion to ln I(f) with a slope [delta T/delta (ln I(f)] of approximately 0.3 s. At higher flash intensities, a different linear relation prevails, in which [deltaT/delta(ln I(f) is approximately 2.3 s [Invest. Ophthalmol. Vis. Sci. 36, 1603 (1995)]. Based on a model for photocurrent recovery in isolated single rods [Vis. Neurosci. 8, 9 (1992)], it was suggested that the upper-branch slope of approximately 2.3 s represents tau R*, the lifetime of photoactivated rhodopsin (R*). Here we show that a modified version of this model provides an explanation for the lower branch of the alpha-wave saturation function. In this model, tau E* is the exponential lifetime of an activated species (E*) within the transducin or guanosine 3', 5'-cyclic monophosphate (cGMP) phosphodiesterase stages of rod phototransduction; the generation of E* by a single R* occurs within temporally defined, elemental domains of disk membrane; and Ex, the immediate product of E* deactivation, is converted only slowly (time constant tau Ex) to E, the form susceptible to reactivation by R*. The model predicts that the decay of flash-activated cGMP phosphodiesterase (PDE*) is largely independent of the deactivation kinetics of R* at early postflash times (i.e., at times preceding or comparable with the lifetime tau E*) and that the lower-branch slope (approximately 0.3s) of the a-wave saturation function represent tau E*. The predicted early-stage independence of PDE* decay and R* deactivation furthermore suggests a basis for the relative constancy of the single-photon response observed in studies of isolated rods. Numerical evaluation of the model yields a value of approximately 6.7s for the time constant tau Ex.

Increased respiratory burst and phosphodiesterase activity in alveolar eosinophils in chronic eosinophilic pneumonia

A 43 year old woman presented with chronic eosinophilic pneumonia characterised by a high alveolar eosinophilic count, which allowed biochemical study of these cells. Alveolar eosinophils spontaneously produced high amounts of oxygen free radicals and exhibited an increased level of cyclic adenosine monophosphate (cAMP) phosphodiesterase (PDE) activity compared to blood eosinophils from control or allergic subjects. This activity was preferentially located in the plasma membrane, whilst the PDE activity of blood eosinophils from asthmatics or controls predominated in the cytosol. Because of the potential role of phosphodiesterase during eosinophil activation and recruitment, phosphodiesterase inhibitors may be useful in the treatment of eosinophilic pneumonia.

Suppression of oro-facial movements by rolipram, a cAMP phosphodiesterase inhibitor, in rats chronically treated with haloperidol

We investigated the effects of rolipram, a selective cyclic adenosine 3′,5′-monophosphate phosphodiesterase type IV inhibitor, and isobutylmethylxanthine, a nonselective phosphodiesterase inhibitor, on purposeless spontaneous chewing movements and tongue protrusions produced by 24 weeks treatment with haloperidol decanoate (25 mg/kg every 4 weeks i.m.) in rats, to examine our hypothesis that restoration of striatal cyclic adenosine 3′,5′-monophosphate levels previously reduced due to dopamine D 2 receptor supersensitivity, may suppress these movements. Tests were performed 8 weeks after the final injection. Haloperidol treatment significantly increased dyskinetic movements and striatal dopamine D 2 receptor density compared with controls. Rolipram (0.1–1.0 mg/kg i.p.) suppressed these movements in a dose-dependent manner, whereas isobutylmethylxanthine (2 mg/kg i.p.) only slightly suppressed the syndrome and doses higher than 5 mg/kg i.p. produced other intensive movements. These results support our hypothesis and suggest that rolipram may have a therapeutic effect on tardive dyskinesia.

Determination of a cyclic guanine monophosphate phosphodiesterase inhibitor (SCH 51866) in rat serum using capillary zone electrophoresis.

A capillary zone electrophoretic (CZE) assay was developed for the determination of cis-5,6a,7,8,9,9a-hexahydro-2-[4-(trifluoromethyl)phenylmethyl]-5-methyl - cyclopent[4,5]imidazo[2,1-b]purin-4(3H)-one, SCH 51866 (I), a cyclic guanine monophosphate phosphodiesterase inhibitor, in rat serum using acetonitrile deproteination as a clean-up step before injection. The calibration curve was linear over a serum concentration range of 0.5 to 10 micrograms/ml serum with a correlation coefficient (r) > 0.99. The limit of quantitation (LOQ) was established at 0.5 micrograms/ml. Fifty microliters of serum were used for analysis, which allowed serial bleeding (8 samples) from a single rat to characterize the pharmacokinetic profile of I after either oral or intravenous drug administration. In traditional pharmacokinetic and toxicokinetic studies in rodents, one animal provides only one serum sample since 1 to 2 ml of sample volume is required for chromatographic analysis, resulting in the use of a large number of animals per study. This assay yields a significant reduction in the use of animals, hence providing a large reduction in resources and time in drug discovery and development.

Porcine detrusor cyclic nucleotide phosphodiesterase isoenzymes: Characterization and functional effects of various phosphodiesterase inhibitors in vitro

Objectives This study was undertaken to characterize adenosine 3′5′cyclic monophosphate (cAMP) and guanosine 3′5′-cyclic monophosphate (cGMP) phosphodiesterases (PDEs) in porcine detrusor smooth muscle and to define their possible role in tension regulation. Methods PDEs were isolated from porcine detrusor homogenate by Q-Sepharose an ion exchange and calmodulin affinity chromatography. The effects of selective inhibitors of CAMP and cCMP PDEs were investigated on isolated PDEs and on carbachol (1 μM) precontracted detrusor strips. Results Six PDE isoenzymes were isolated by Q-Sepharose anion exchange and calmodulin affinity chromatography: one calmodulin-stimulated PDE (PDE I) which hydrolyzed mainly cGMP, one cGMP-stimulated cAMP PDE (PDE II), two cAMP-specific PDE (PDE IVα and IVβ), and two cGMP-specific PDE (PDE Vα and Vβ). PDE I was potently inhibited in a dose-dependent fashion by papaverine, vinpocetine, and zaprinast; the PDE IVs were potently inhibited by papaverine and rolipram; and the PDE Vs were weakly inhibited by papaverine. In organ bath studies, inhibitors of PDE III (milrinone), IV (rolipram), and V (zaprinast) caused only minor relaxations at high concentrations (200 μM), whereas papaverine and vinpocetine caused relaxations of more than 50%. Conclusions Our findings support the involvement of cyclic nucleotide metabolism in the regulation of the detrusor smooth muscle tone in the pig and its regulation by PDEs. The weak action of PDE IV and V inhibitors in vitro may be explained by a possible intracellular compartmentalization of such PDEs and the low cyclic nucleotide turnover rate at the conditions used.

Activation of protein kinase C stimulates cAMP phosphodiesterase in rat renal collecting tubule.

The present study was undertaken to evaluate whether protein kinase C (PKC) affects adenosine 3',5'-cyclic monophosphate phosphodiesterase (cAMP-PDIE) activity in microdissected rat renal medullary collecting tubules (MCT). Phorbol 12-myristate 13-acetate (PMA, 10(-8) M), an activator of PKC, significantly stimulated cAMP-PDIE activity in intact MCT (29.5 +/- 1.5 to 38.3 +/- 3.7 fmol.min-1.mm-1, P < 0.05) but not in the proximal straight tubule [4.7 +/- 0.8 vs. 4.8 +/- 0.8, not significant (NS)] or the medullary ascending limb of Henle's loop (20.5 +/- 2.1 vs. 22.4 +/- 2.8, NS). PMA-stimulated cAMP-PDIE activity was reversed by PKC inhibitors, staurosporine (10(-8) M) and calphostin C (10(-8) M), but not by the cyclooxygenase inhibitor, indomethacin (5 x 10(-6) M). 1,2-Dioctanoyl-sn-glycerol (50 micrograms/ml), a synthetic analogue of diacylglycerol that stimulates PKC, also increased cAMP-PDIE activity in broken-cell preparations from MCT. This stimulation was also suppressed by staurosporine (10(-8) M) and calphostin C (10(-8) M). The stimulatory effect of PMA on cAMP-PDIE activity was lost with rolipram (10(-4) M), a type IV PDIE inhibitor, whereas it was preserved with N-(6-amino-hexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7) (10(-4) M), a calmodulin inhibitor, or vinpocetin (10(-4) M), a direct inhibitor of type I PDIE. From these results, we suggest that activation of PKC specifically stimulates rolipram-sensitive cAMP-PDIE, but not the calmodulin-sensitive isozyme, in rat MCT.

A novel 5-HT1-like receptor subtype mediates cAMP synthesis in porcine pial vein.

The 5-hydroxytryptamine (5-HT) receptor subtype mediating 5-HT inhibition of spontaneous rhythmic contractions (SRC) in the porcine pial vein was characterized. Results from pharmacological studies using in vitro tissue bath techniques indicated that the inhibitory effects of 5-HT on SRC were qualitatively and quantitatively mimicked by 5-HT1-like agonists 5-methoxytryptamine (5-MT) and 5-carboxamidotryptamine (5-CT). 5-HT-, 5-MT-, and 5-CT-induced inhibitions of SRC were attenuated in a concentration-dependent manner by methysergide, which yielded similar pA2 values against these three agonists, suggesting that 5-HT, 5-MT, and 5-CT act on the same 5-HT1-like receptors. 5-MT inhibition of SRC was not affected by blocking 5-HT2 (with ketanserin and spiperone), 5-HT3 (with MDL-72222 and ICS-205-930), or 5-HT4 (with ICS-205-930) receptors. Neither was 5-MT inhibition of SRC affected by blocking 5-HT1A (with propranolol and spiperone), 5-HT1B (with propranolol), or 5-HT1C (with ketanserin) receptors. Furthermore, 5-HT and 5-MT inhibitions of SRC were enhanced by cilostazol [a selective adenosine 3',5'-cyclic monophosphate (cAMP) phosphodiesterase inhibitor] and were diminished by KT-5720 (a cAMP-dependent protein kinase inhibitor) but were not affected by M&B-22948 [a selective guanosine 3',5'-cyclic monophosphate (cGMP) phosphodiesterase inhibitor] or KT-5823 (a cGMP-dependent protein kinase inhibitor). Biochemical studies further demonstrated that 5-HT inhibition of SRC in porcine pial veins was accompanied by an increase in cAMP, but not cGMP, synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of a hormone-inducible, high affinity adenosine 3'-5'-cyclic monophosphate phosphodiesterase from the rat Sertoli cell.

In previous reports we have shown that FSH and beta-adrenergic agonists regulate the levels of mRNA and increase the activity of a high affinity cAMP phosphodiesterase (cAMP-PDE) in the immature rat Sertoli cell in culture. To identify and characterize the hormone-inducible form(s), the cAMP-PDE activity of the Sertoli cell was partially purified and its properties were determined using biochemical and immunological tools. The cAMP-PDE activity present in the 100,000g supernatant of Sertoli cell extracts was purified more than 2000-fold by four HPLC chromatographic steps. The major purified form of cAMP-PDE had a specific activity of 1-2 mumol/(min.mg of protein). Polyacrylamide gel electrophoresis and silver staining analysis showed that a 67-68 kDa polypeptide comigrated with the major peak of cAMP hydrolytic activity. The molecular weight of the crude or purified enzyme determined by gel filtration and sucrose density gradients was 150,000, suggesting that the native enzyme is an oligomeric structure. This PDE hydrolyzed cAMP with a Km of 1.97 +/- 0.26 microM. The hydrolysis of cAMP was neither inhibited nor stimulated by cGMP concentrations lower than 50 microM. Cyclic nucleotide catalysis required Mg2+, but was insensitive to Ca2+. The activity of this form was competitively inhibited by several inhibitors with the following potency: rolipram > RO 20-1724 > methylisobutylxanthine > cilostamide = milrinone. Because mRNAs derived from two distinct PDE4B and PDE4D genes are present in the Sertoli cell, selective and nonselective PDE antibodies were used to determine the origin of the inducible PDE protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of mevalonate in smooth muscle cell proliferation

It has long been discussed why dihydropyridine calcium antagonists are less cardioactive than other calcium antagonists at doses equally effective on vascular smooth muscle [5, 12]. Recently, it has been reported that dihydropyridine calcium antagonists, unlike other calcium antagonists, inhibit adenosine 3′,5′-cyclic monophosphate phosphodiesterase in various tissue preparations [2, 8, 10]. It has also been reported that intracellularly applied adenosine 3′,5′-cyclic monophosphate increases the slow inward current in heart cells [1, 11, 13]. Therefore, present experiments were carried out to elucidate the effects of intracellularly applied calcium antagonists on the slow inward current of single ventricular cells with the patchclamp, ‘whole-cell recording’, technique [3]. Nifedipine, nicardipine and isobutylmethylxanthine, when applied intracellularly using the patch-clamp microelectrode, increased the plateau and the duration of action potentials of single ventricular heart cells of the guinea-pig. The augmentation of the action potential was abolished by superfusing the cell with nifedipine. D600 and diltiazem when applied either intra- or extra-cellularly decreased the plateau and the duration of the action potential. Under voltage clamp conditions, intracellularly applied nifedipine, nicardipine and isobutylmethylxanthine increased the slow inward current, whereas D600 and diltiazem decreased the current. The holding current and the current at the end of 200-ms clamp pulses were not modified. Thus, dihydropyridine calcium antagonists probably increase the slow inward current from the inside of the cell and attenuate the calcium entry blocking activities in heart cells. This must partly be a reason why dihydropyridine calcium antagonists are less cardioactive than D600 and diltiazem at doses equally effective on vascular smooth muscle.

Elevated cyclic adenosine monophosphate phosphodiesterase activity in peripheral blood mononuclear leucocytes from children with atopic dermatitis

We examined peripheral blood mononuclear leucocyte cyclic adenosine monophosphate phosphodiesterase (cAMP-PDE) activity in 80 children (aged 2-12 years) with atopic dermatitis. The enzyme activity (35.1 +/- 18.6 U) in children with atopic dermatitis was significantly higher than that (19.1 +/- 12.6 U) in age-matched non-atopic controls. There was no significant difference in the cAMP-PDE activity between children with mild atopic dermatitis and children with severe atopic dermatitis. These findings support the view that elevation of peripheral mononuclear leucocyte cAMP-PDE activity in patients with atopic dermatitis is a gene-associated abnormality.

Age-dependent changes in second messenger and rolipram receptor systems in the gerbil brain.

Age-related alterations in binding sites of major second messengers and a selective adenosine 3',5'-cyclic monophosphate (cyclic-AMP) phosphodiesterase (PDE) in the gerbil brain were analysed by receptor autoradiography. [3H]Phorbol 12,13-dibutyrate (PDBu), [3H]inositol 1,4,5-trisphosphate (IP3), [3H]forskolin, [3H]cyclic-AMP, and [3H]rolipram were used to label protein kinase C (PKC), IP3 receptor, adenylate cyclase, cyclic-AMP dependent protein kinase (PKA), and Ca2+/calmodulin-independent cyclic-AMP PDE, respectively. In middle-aged gerbils (16 months old), [3H]PDBu binding was significantly reduced in the hippocampal CA1 sector, thalamus, substantia nigra, and cerebellum, compared with young animals (1 month old). [3H]IP3 binding revealed significant elevations in the nucleus accumbens, hippocampal CA1 sector, dentate gyrus, and a significant reduction in cerebellum of middle-aged gerbils. [3H]Forskolin binding in middle-aged animals was significantly increased in the nucleus accumbens and hilus of dentate gyrus, but was diminished in the substantia nigra and cerebellum. On the other hand, in middle-aged animals, [3H]cyclic-AMP binding revealed a significant elevation only in the hippocampal CA3 sector, whereas [3H]rolipram binding showed a significant reduction in the thalamus and cerebellum. Thus, the age-related alteration in these binding sites showed different patterns among various brain regions in middle-aged gerbils indicating that the binding sites of PKC, IP3, and adenylate cyclase are more markedly affected by aging than those of PKA and cyclic-AMP PDE and that the hippocampus and cerebellum are more susceptible to these aging processes than other brain regions. The findings suggest that intracellular signal transduction is affected at an early stage of senescence and this may lead to neurological deficits.

Analysis of responses to bradykinin in the pulmonary vascular bed of the cat.

Responses to bradykinin (BK) were investigated in the pulmonary vascular bed of the cat under conditions of controlled pulmonary blood flow and constant left atrial pressure when lobar arterial pressure was elevated to a high steady level. Under elevated-tone conditions, BK caused dose-related decreases in lobar arterial pressure. After administration of Hoe-140, a BK B2-receptor antagonist, vasodilator responses to BK were reduced in a selective manner. Vasodilator responses to BK were unchanged by atropine, glibenclamide, meclofenamate, or bronchial occlusion, suggesting that responses are not dependent on the activation of muscarinic receptors or K+ATP channels, the release of vasodilator prostaglandins, or changes in bronchomotor tone. The nitric oxide (NO) synthase inhibitors N omega-nitro-L-arginine benzyl ester and N omega-nitro-L-arginine reduced vasodilator responses to BK in a selective manner, indicating that responses to BK are mediated in part by the release of NO. Methylene blue, an inhibitor of the activation of soluble guanylate cyclase, increased lobar arterial pressure and decreased responses to BK. The increases in lobar arterial pressure in response to methylene blue were partially reversed by the administration of superoxide dismutase, indicating that generation of O2- may inactivate basally released NO. The duration of the response to BK was enhanced by the guanosine 3',5'-cyclic monophosphate (cGMP) phosphodiesterase inhibitor Zaprinast, suggesting that responses to BK involve increases in cGMP levels. Responses to BK were enhanced by captopril, indicating that BK is rapidly inactivated by kininase II in the lung.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphodiesterase inhibitors. Do the risks outweight the benefits?

Despite the justifiable concern about the use of oral cyclic adenosine monophosphate phosphodiesterase (cAMP PDE) inhibitors, the intravenous preparations are clearly effective in the treatment of acute heart failure, whether it occurs de novo or complicating chronic congestive heart failure (CHF), and in low output states following cardiac surgery. There are no grounds to curtail their use in these areas, though any advantages over conventional agents such as dobutamine need further investigation with regard to end-points other than haemodynamic parameters. The long term use of oral cAMP PDE inhibitors in the treatment of chronic CHF should remain restricted by the increase in mortality now confirmed in severe CHF. However, in view of the distressing nature of the condition there remain subgroups of patients in whom the benefits may outweight the risks.

Atrial Natriuretic Factor Activates Cyclic Adenosine 3′,5′-Monophosphate Phosphodiesterase in Xenopus Laevis Oocytes and Potentiates Progesterone-Induced Maturation via Cyclic Guanosine 5′-Monophosphate Accumulation

Xenopus oocytes were found to express atrial natriuretic factor (ANF) receptors that activate guanylate cyclase and stimulate cyclic guanosine 5'-monophosphate (cGMP) production in a dose- and time-dependent manner. A truncated fragment of ANF, known to bind to mammalian ANF receptors without stimulating cGMP accumulation, did not elicit a cGMP response in oocytes. In addition, preincubation with ANF increased the number of oocytes that underwent progesterone-induced maturation. The maximally effective dose of ANF (1 microM) elevated intracellular and extracellular cGMP accumulation from 50 to 200 and 5 to 800 fmol/oocyte, respectively, and increased the number of maturing oocytes by up to 3-fold. ANF's effects on progesterone-induced maturation were mimicked by nonhydrolyzable analogues of cGMP. ANF and 8-Br-cGMP had no effect on maturation in the absence of progesterone, indicating that elevation of cGMP alone is not sufficient to induce maturation. Dibutyryl-cGMP was as effective as 8-Br-cGMP, whereas 8-Br-guanosine monophosphate, 8-Br-guanosine, and 8-Br-cyclic inosine monophosphate did not potentiate ovum maturation. In Xenopus oocytes, an initial step in progesterone-induced maturation is the reduction of intracellular cAMP levels; both ANF and 8-Br-cGMP lowered cAMP levels and enhanced progesterone's ability to do so. This decrease in cAMP levels was attributable to increased cAMP-phosphodiesterase activity, which was enhanced by both ANF and 8-Br-cGMP. These findings, and the presence of functional ANF receptors on Xenopus oocytes, demonstrate that ANF can participate in ovum development by stimulation of cGMP accumulation and activation of cAMP phosphodiesterase, thereby potentiating progesterone's ability to decrease cAMP levels and promote ovum maturation.

Appearance of cGMP-phosphodiesterase immunoreactivity parallels the morphological differentiation of photoreceptor outer segments in the rat retina.

We have investigated by immunofluorescence the appearance of immunoreactive guanosine 3'-5' cyclic monophosphate phosphodiesterase (cGMP-PDE) during the postnatal development of the retina of the pigmented rat. We show that a sudden increase in immunoreactivity takes place during postnatal day five (P5), when rod outer segments begin to form; immunoreactivity develops rapidly in the following days. Labeling is restricted to the developing photoreceptor outer segments, sparing other retinal cells, as confirmed by electron microscopy immunocytochemistry. In addition, cGMP-PDE immunoreactivity follows a center-to-periphery gradient paralleling photoreceptor differentiation. It appears that cGMP-PDE is expressed when the photoreceptor subcellular compartments are already formed, and represents a specific marker of late photoreceptor differentiation. The appearance of cGMP-PDE during development is temporally correlated with the appearance of other proteins of the phototransduction machinery.

Peruvianosides A and B, Novel Triterpene Glycosides from the Bulbs of Scilla peruviana

Abstract Two new rearranged lanosterol trisaccharides, peruvianosides A (1) and B (2), were isolated from fresh bulbs of Scilla peruviana. Their structures were established mainly by extensive 2D NMR analysis. Peruvianoside A (1) showed an inhibitory activity on cyclic adenosine monophosphate (AMP) phosphodiesterase.

The effect of cyclic AMP and cyclic GMP phosphodiesterase inhibitors on the superoxide burst of guinea‐pig peritoneal macrophages

1. The cyclic nucleotide phosphodiesterase (PDE) activity of guinea-pig peritoneal macrophages was partially characterized and the effects of selective and non-selective inhibitors of adenosine 3':5'-cyclic monophosphate (cyclic AMP PDE) and guanosine 3':5'-cyclic monophosphate (cyclic GMP PDE) phosphodiesterases on superoxide generation were investigated using peritoneal macrophages from horse-serum pretreated guinea-pigs. 2. The non-selective PDE inhibitor, 3-isobutyl-1-methylxanthine (IBMX) and the PDE I/V selective inhibitor, zaprinast, inhibited spontaneous superoxide generation with IC50s of 30.7 +/- 11.3 microM and 145 +/- 17 microM respectively (n = 6 and 5). The concentration-response curves for the PDE IV selective inhibitors rolipram and Ro20-1724 were biphasic; mean maximum inhibitions were 56.9 +/- 5.9% and 66.8 +/- 10.5% respectively at 300 microM, but in 2 out of 6 (rolipram) and 2 out of 5 (Ro20-1724) experiments inhibition was < 50%. The PDE III inhibitor SK&F 94120 was without effect. Spontaneous superoxide generation was reduced 57 +/- 10% by 1 microM prostaglandin E2 (PGE2) and 62.6 +/- 3.76% by 1 microM salbutamol. 3. The increase in superoxide generation elicited by FMLP (10(-9)-10(-5)M) was unaffected by any of the PDE inhibitors studied. Inhibition of FMLP-stimulated superoxide generation by PGE2 was enhanced in the presence of 10 microM IBMX. 4. Macrophages were found to contain a predominantly membrane bound cyclic AMP PDE (90% of total activity) which was unaffected by cyclic GMP or calcium/calmodulin. The cyclic AMP PDE activity in the cytosolic fraction was enhanced in the presence of calcium/calmodulin. Selective inhibitors of PDE IV inhibited the particulate cyclic AMP PDE activity (IC50s rolipram 1.5 +/- 0.3 microM, Ro 20-17244.1 +/- 0.6 microm) as did the non-selective inhibitor IBMX (IC50 22 +/- 8 microM). The macrophage particulate PDE activity was resistant to inhibition by the PDE III inhibitor SK&F 94836 and the PDE I/V inhibitor, zaprinast. The cytosolic calcium/calmodulin stimulated cyclic AMP hydrolytic activity was inhibited by zaprinast (IC50 - calcium/calmodulin 123 +/- 39 microM; + calcium/calmodulin IC50 17.7 +/- 6.3 microM).5. The results indicate that guinea-pig peritoneal macrophages contain a type IV cyclic AMP PDE which is predominantly membrane associated and a predominantly cytosolic calcium/calmodulin stimulated cyclic AMP PDE. Functional studies suggest that both of these PDE activities contribute to cyclic AMP hydrolysis and regulation of superoxide generation in these cells. Inhibition of spontaneous superoxide generation, but not that stimulated by FMLP, suggests that the activity of PDE inhibitors is subject to functional antagonism but that this can be overcome by enhancing cyclic AMP formation.

Lack of cross-tolerance between nitroglycerin and endothelium-derived relaxing factor-mediated vasoactive agents in spontaneously hypertensive rats

The purpose of this study was to determine whether cross-tolerance develops between nitroglycerin and endothelium-derived relaxing factor (EDRF)-mediated vasoactive agents in vivo. Spontaneously hypertensive rats (SHR) were made tolerant by pretreatment with high doses of nitroglycerin (100 mg/kg s.c., 3 times/day, for 3 consecutive days). The hypotensive effect of challenge doses of nitroglycerin (1, 10, 300, 100 μg/kg i.v.) was completely abolished in nitroglycerin-pretreated SHR. To evaluate cross-tolerance, the effects of the following EDRF-dependent vasoactive agents on blood pressure were determined in groups of nitroglycerin-pretreated and vehicle-pretreated SHR: acetylcholine, bradykinin and L-arginine. In addition, the hypotensive effects of zaprinast (M&B 22,928), a cyclic guanosine monophosphate (cGMP) phosphodiesterase inhibitor, and the hypertensive effects of the nitric oxide-synthase inhibitor N ω-nitro-L-arginine were also evaluated. In all cases, there was no difference in the effects of these agents on blood pressure when compared in nitroglycerin-pretreated (tolerant) and vehicle-pretreated (non-tolerant) SHR. The use of a variety of agents which modulate EDRF release or its effects by several different mechanisms suggests that cross-tolerance does not occur between nitroglycerin and EDRF in vivo.