Cyclic Nucleotide Phosphodiesterase Isoforms Research Articles

GENIe: A bright new genetically‐encoded fluorescent biosensor for monitoring cGMP

GENIe is a Genetically Encoded Nucleotide Indicator for monitoring cGMP, a second messenger in cell signaling that mediates many important physiological processes. Phosphodiesterase (PDE) inhibitors and soluble guanylate cyclase (sGC) activators modulate cGMP levels to produce beneficial effects on pulmonary and heart disease, erectile dysfunction, and other conditions. The timing, location and strength of cGMP signaling is tightly regulated by nearly 100 isoforms of cyclic nucleotide PDEs (Bender and Bravo 2006). Some isoforms are specific for cGMP, while others are specific for cAMP. Some are nonspecific and act on both cAMP and cGMP.We have developed a new, very bright mNeonGreen fluorescent biosensor to monitor cGMP levels in any living cell type. The sensor produces robust changes in fluorescence intensity that are readily detected on fluorescence imaging systems or plate readers. Since different PDE isoforms have different substrate specificities, we did parallel assays of cGMP and cAMP in HEK293 cells expressing either the cGMP biosensor (GENIe) or the cAMP biosensor (cADDis). These sensors produced markedly different fluorescent profiles of cGMP and cAMP responses when treated with different PDE inhibitors. Our results indicate that the combination of these assays provide useful insights into the specific action of a particular PDE inhibitor in a living cell. Since these sensors can be genetically targeted, and since many biologically relevant cell types express more than one PDE, these tools provide a new approach to understanding the specific action of PDE inhibitors in health and disease.We tested GENIe in HEK293T cells on a BioTek Synergy MX automated fluorescence plate reader. cGMP production was initiated by adding sodium nitroprusside (SNP), a nitric oxide donor, in both the presence and absence of the PDE5 selective inhibitor, Sildenafil. Fluorescence intensity changed dramatically in the presence of Sildenafil, as degradation of cGMP is inhibited by the drug. To optimize the response in HEK293T cells, we over‐expressed sGC, treated with SNP, and observed a significant improvement in the amplitude of the signal accompanied by faster kinetics and an increased sensitivity to the nitric oxide donor SNP. We expect that endogenous levels of sGC will vary in different cell types and these parameters should be varied accordingly.To examine PDE inhibitor specificity, we monitored responses from both cADDis and GENIe in the presence of different PDE inhibitors. IBMX, a non‐specific inhibitor, produced similar responses from both sensors. Sildenafil and Rolipram indicated cGMP vs cAMP specificity. Sildenafil exhibited selective inhibition of the cGMP‐specific PDE5, while Rolipram exhibited selective inhibition of the cAMP specific PDE4.Support or Funding InformationFunding from NSF SBIR Phase IIB 1430878This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cyclic nucleotide phosphodiesterase isoforms in human basophils and mast cells.

Cyclic nucleotide phosphodiesterase (PDE) exists as multiple molecular forms. Of the 11 families of PDE identified so far, PDE4, a cAMP-specific PDE, has been identified as the major isoform regulating inflammatory activity. The principle aim of the present study was to determine whether human basophils and human lung mast cells express PDE4. Four sub-classes of PDE4 (A, B, C, and D) have been identified and expression of these was determined by RT-CPR and by western blotting. In basophils, prominent expression of mRNA for PDE4A and PDE4D was observed whereas little if any expression of PDE4B and PDE4C was detected. These findings were paralleled by immunoblotting experiments as human basophils were found to express PDE4A and PDE4D with little evidence for the presence of either PDE4B or PDE4C. By contrast, human lung mast cells expressed very little, if any, mRNA for PDE4 sub-classes although, in some preparations, some modest levels of mRNA for PDE4D were detected. However, there was no evidence, at the protein level, that mast cells express PE4. Overall, these data indicate that basophils express PDE4 (4A and 4D) whereas human lung mast cells do not.

The flavonoid dioclein is a selective inhibitor of cyclic nucleotide phosphodiesterase type 1 (PDE1) and a cGMP-dependent protein kinase (PKG) vasorelaxant in human vascular tissue

The inhibitory effect of the flavonoid dioclein was assessed on purified vascular cyclic nucleotide phosphodiesterase isoforms (EC 3.1.4.17, PDE1-5) in comparison with 8-methoxymethyl-isobutylmethylxanthine (8-MM-IBMX) and vinpocetine which are currently used as PDE1 inhibitors. The mechanism underlying the vasorelaxant effect of dioclein was investigated in human saphenous vein. Dioclein inhibited PDE1 more selectively than vinpocetine and 8-MM-IBMX, with IC 50 values of 2.47 ± 0.26 and 1.44 ± 0.35 µM, respectively in basal- and calmodulin-activated states. Dioclein behaved as a competitive inhibitor for cGMP hydrolysis by PDE1 in basal- and calmodulin-activated states ( K i = 0.62 ± 0.14 and 0.55 ± 0.07 µM, respectively), indicating this inhibitory effect to be independent of calmodulin interactions. In addition, dioclein induced a concentration-dependent relaxation of human saphenous vein which was independent on the presence of functional endothelium (EC 50 values of 7.3 ± 3.1 and 11 ± 2.7 µM, respectively with and without endothelium). 8-MM-IBMX relaxed human saphenous vein with an EC 50 = 31 ± 16 µM, whereas vinpocetine did not cause any vasorelaxation at concentrations up to 100 µM. Rp-8-pCPT-cGMPS, which inhibits cGMP-dependent protein kinase (PKG), blocked the vasodilator effect of dioclein, whereas H-89, which is a cAMP-dependent protein kinase (PKA) inhibitor, had a minor inhibitory effect. Our data show that dioclein is a potent calmodulin-independent selective inhibitor of PDE1 and that inhibition of PDE1 is involved in the PKG-mediated vasorelaxant effect of dioclein in human saphenous vein. Furthermore, dioclein may represent a new archetype to develop more specific PDE1 inhibitors.

Role of cyclic nucleotide phosphodiesterase isoforms in cAMP compartmentation following beta2-adrenergic stimulation of ICa,L in frog ventricular myocytes.

The role of cyclic nucleotide phosphodiesterase (PDE) isoforms in the beta2-adrenergic stimulation of the L-type Ca2+ current (ICa,L) was investigated in frog ventricular myocytes using double patch-clamp and double-barrelled microperfusion techniques. Isoprenaline (ISO, 1 nM to 10 microM) was applied on one half of the cell, either alone or in the presence of PDE inhibitors, and the local and distant responses of ICa,L were used to determine the gradient of local vs. distant cAMP concentration (alpha). IBMX (100 microM), a non-selective PDE inhibitor, reduced alpha from 40 to 4.4 indicating a 9-fold reduction in intracellular cAMP compartmentation when all PDE activity was blocked. While PDE1 and PDE2 inhibition had no effect, PDE3 inhibition by milrinone (3 microM) or PDE4 inhibition by Ro 20-1724 (3 microM) reduced alpha by 6- and 4-fold, respectively. A simultaneous application of milrinone and Ro 20-1724 produced a similar effect to IBMX, showing that PDE3 and PDE4 were the major PDEs accounting for cAMP compartmentation. Okadaic acid (3 microM), a non-selective phosphatase inhibitor, or H89 (1 microM), an inhibitor of cAMP-dependent protein kinase (PKA), had no effect on the distant response of ICa,L to ISO indicating that PDE activation by PKA played a minor role in cAMP compartmentation. Our results demonstrate that PDE activity determines the degree of cAMP compartmentation in frog ventricular cells upon beta2-adrenergic stimulation. PDE3 and PDE4 subtypes play a major role in this process, and contribute equally to ensure a functional coupling of beta2-adrenergic receptors with nearby Ca2+ channels via local elevations of cAMP.

Role of cyclic nucleotide phosphodiesterase isoforms in cAMP compartmentation following beta2-adrenergic stimulation of ICa,L in frog ventricular myocytes

The role of cyclic nucleotide phosphodiesterase (PDE) isoforms in the β2-adrenergic stimulation of the L-type Ca2+ current (ICa,L) was investigated in frog ventricular myocytes using double patch-clamp and double-barrelled microperfusion techniques. Isoprenaline (ISO, 1 nM to 10 μM) was applied on one half of the cell, either alone or in the presence of PDE inhibitors, and the local and distant responses of ICa,L were used to determine the gradient of local vs. distant cAMP concentration (α). IBMX (100 μM), a non-selective PDE inhibitor, reduced α from 40 to 4.4 indicating a 9-fold reduction in intracellular cAMP compartmentation when all PDE activity was blocked. While PDE1 and PDE2 inhibition had no effect, PDE3 inhibition by milrinone (3 μM) or PDE4 inhibition by Ro 20-1724 (3 μM) reduced α by 6- and 4-fold, respectively. A simultaneous application of milrinone and Ro 20-1724 produced a similar effect to IBMX, showing that PDE3 and PDE4 were the major PDEs accounting for cAMP compartmentation. Okadaic acid (3 μM), a non-selective phosphatase inhibitor, or H89 (1 μM), an inhibitor of cAMP-dependent protein kinase (PKA), had no effect on the distant response of ICa,L to ISO indicating that PDE activation by PKA played a minor role in cAMP compartmentation. Our results demonstrate that PDE activity determines the degree of cAMP compartmentation in frog ventricular cells upon β2-adrenergic stimulation. PDE3 and PDE4 subtypes play a major role in this process, and contribute equally to ensure a functional coupling of β2-adrenergic receptors with nearby Ca2+ channels via local elevations of cAMP.

Characterisation of cyclic nucleotide phosphodiesterase isoforms in the media layer of the main pulmonary artery

Cyclic nucleotides are involved in the control of pulmonary vascular tone. In the present study, we measured the cyclic nucleotide specific phosphodiesterase (PDE) activity in the media of bovine isolated main pulmonary artery (MPA). Total cAMP- and cGMP-PDE activities were measured in microsomal and cytosolic fractions. Both cyclic nucleotides were hydrolysed in these subcellular fractions at consistently higher rate in the cytosolic than in the microsomal fraction. Using different classes of PDE modulator, at least four PDE isoforms (PDE1, 3, 4 and 5) were identified in these fractions. PDE3 (cilostamide-sensitive), PDE4 (rolipram-sensitive) and PDE5 (zaprinast- and DMPPO-sensitive) isoforms appeared as the main isozymes implicated in the cAMP and cGMP hydrolytic activities. Calcium–camodulin stimulated PDE activity (PDE1) was mainly present in the cytosolic fraction. PDE2, although present, had a lower hydrolytic activity since addition of its specific inhibitor, erythro-9-(2-hydroxy-3nonyl)adenine (EHNA), to a combination of inhibitors of PDE3, 4 and 5 produced no further significant reduction in the enzymatic activity. Resolution of PDE activities from the cytosolic fraction using anion exchange chromatography confirmed this finding. Functional experiments performed in endothelium-denuded rings of rat MPA revealed that all specific PDE inhibitors used relaxed precontracted vascular smooth muscle preparations in a concentration-dependent manner. The rank order of potency was cilostamide >zaprinast>rolipram>>EHNA. The present study demonstrates the presence in the smooth muscle cells-containing layer of MPA of PDE1, 3, 4 and 5 isoforms and suggests that PDE3, 4 and 5 are the main enzymes involved in the control of vascular tone.

Role of cyclic nucleotide phosphodiesterase isoenzymes in contractile responses of denuded rat aorta related to various Ca2+ sources.

We have examined the cyclic nucleotide phosphodiesterase isoforms (PDE) involved in the contractile response of rat aorta to different agonists and different experimental procedures for use in functional studies. The inhibitory effect of AAL 05 on the different PDEs isolated from bovine aortic smooth muscle was examined. Compound AAL 05 appeared to be a selective PDE3 inhibitor. We analyzed the ability of the non-selective inhibitor IBMX (3-isobutyl-1-methylxanthine) and the isoenzyme selective inhibitors nimodipine (type 1), AAL 05 (6-(N-methyl-N-cyclohexyl butyl carboxamide) quinolin-2-one) and SK&F 94120 (5-(4-acetamidophenyl) pyrazin-2(1H)-one; type3), rolipram (type4) and zaprinast (type5) to affect the contractile responses of denuded rat aortic rings to KCl (80 mM) and noradrenaline (NA, 1 microM) in the presence or absence of extracellular Ca2+. Rolipram (10-100 microM) and zaprinast (1-100 microM) failed to relax the aortic strips, but IBMX (0.1-30 microM), nimodipine (1 fM10 microM), AAL 05 (0.01-100 microM) and SK&F 94120 (0.1-100 microM) produced a concentration-dependent relaxation or inhibition of contractile responses to the different agonists, but the pIC50 obtained for each inhibitor was different depending on the experimental procedure. Except for nimodipine (a Ca2+ channel blocker), all the PDE inhibitors showed the following rank of potency: pIC50 on NA-induced contractions in Ca2+-free medium > pIC50 on NA-induced contractions in Ca2+-containing solution > pIC50 on depolarizing solution-induced contraction. This ranking apparently depends on the differences in the Ca2+ sources. We obtained a good correlation between the pKi of PDE3 inhibitors in biochemical studies and the pIC50 on NA-induced contraction in Ca2+-free medium. In conclusion, PDE1 and PDE3 isoenzymes play an important role as modulators of rat aortic smooth muscle contractility regardless of the experimental procedure used. Since intracellular mechanisms are more dependent on PDE activity, experimental procedures performed in absence of extracellular calcium are the most suitable for analyzing the modulatory role of PDE inhibitors.

Differential changes in the expression of cyclic nucleotide phosphodiesterase isoforms in rat brains by chronic treatment with electroconvulsive shock.

Electroconvulsive shock (ECS) has been suggested to affect cAMP signaling pathways to exert therapeutic effects. ECS was recently reported to increase the expression of PDE4 isoforms in rat brain, however, these studies were limited to PDE4 family in the cerebral cortex and hippocampus. Thus, for comprehensive understanding of how ECS regulates PDE activity, the present study was performed to determine whether chronic ECS treatment induces differential changes in the expression of all the PDE isoforms in rat brains. We analyzed the mRNA expression of PDE isoforms in the rat hippocampus and striatum using reverse transcription polymerase chain reaction. We found chronic ECS treatment induced differential changes in the expression of PDE isoform 1, 2, 3, 4, 5 and 7 at the rat hippocampus and striatum. In the hippocampus, the expression of PDE1A/B (694%), PDE4A (158%), PDE4B (323 %), and PDE4D (181%) isoforms was increased from the controls, but the expression of PDE2 (62.8%) and PDE7 (37.8%) decreased by chronic ECS treatment. In the striatum, the expression of PDE1A/B (179%), PDE4A (223%), PDE4B (171%), and PDE4D (327%) was increased by chronic ECS treatment with the concomitant decrease in the expression of PDE2 (78.4%) and PDE3A (67.1%). In conclusion, chronic ECS treatment induces differential changes in the expression of most PDE isoforms including PDE1, PDE2, PDE3, PDE4, PDE5, and PDE7 in the rat hippocampus and striatum in an isoform- and brain region-specific manner. Such differential change is suggested to play an important role in regulation of the activity of PDE and cAMP system by ECS.

Characterization of cyclic nucleotide phosphodiesterase isoforms associated to isolated cardiac nuclei

The identity and location of nuclear cyclic nucleotide phosphodiesterases (PDE) has yet to be ascertained. Intact cardiac nuclei and subnuclear fractions from ovine hearts were isolated to determine cAMP-specific PDE activity which was 3-fold greater than that of cGMP PDE, the latter being insensitive to Ca-calmodulin and zaprinast. Specific hydrolytic activities of the cardiac nuclear envelopes (NE) were similar to those measured in the corresponding intact nuclei, thus suggesting that most PDE activity is associated with the nuclear membrane. Moreover, the main hydrolytic activities in cardiac nuclei were attributed to PDE4 (56%) and PDE3 (44%). The pharmacological sensitivity of each isoform in terms of IC 50, K m and K i values was typical of previously characterized cardiac PDE 3 and 4 isoforms. PDE2 (cGMP-stimulated PDE) represented a minor component (8–9%) of total hydrolytic activity. Solubilization of nuclear envelopes and HPLC separation also yielded rolipram-sensitive PDE activities. Upon 1% Triton X-100 extractions, the presence of PDE3 and PDE4 was revealed in a low speed, nucleopore complex-enriched, P1 pellet. In addition, Western blot analysis demonstrated the presence of PDE4B and PDE4D subtypes in the nuclei as well as enrichment in NE. However, in the same preparations, the presence of PDE4A could not be ascertained. Altogether, these results suggest an intrinsic and predominant association of these nuclear PDEs with the NE and much likely with nucleopore complexes.

Altered Expression of PDE1 and PDE4 Cyclic Nucleotide Phosphodiesterase Isoforms in 7-oxo-prostacyclin-preconditioned Rat Heart

The stable prostacyclin derivative, 7-oxo-prostacyclin, exhibits a delayed, long-lasting cardioprotective effect, which is accompanied by an increase in cyclic nucleotide phosphodiesterase (PDE) activities restricted to the Ca2+-calmodulin-dependent (PDE1) and cyclic AMP-specific phosphodiesterase (PDE4) activities. Mammalian PDEs form a large multi-gene family with differential expression occurring in a cell- and tissue-specific manner. The aim of this study was to identify which isoforms of PDE1 and PDE4 are present in the hearts of control and 7-oxo-prostacyclin treated rats. Using RT-PCR analysis, we were able to identify in control rat hearts transcripts for PDE1C, but not for either PDE1A or PDE1B within the three-gene PDE1 family. Within the four-gene PDE4 family we detected, by generic RT-PCR analysis, transcripts for PDE4A, PDE4B and PDE4D, but not PDE4C. Using RT-PCR primers for specific splice variants, we identified transcripts for PDE4B1, PDE4B2, PDE4B3, PDE4D1, PDE4D2 and PDE4D3 in hearts from the control animals. Immunoblotting of hearts from the control animals for PDE4 forms allowed us to identify a 98-kDa PDE4A species, 68-kDa band representing PDE4D1/2 and a 95-kDa species indicative of PDE4D3. In the hearts of treated animals, 48 h after a single 50μg/kg dose of 7-oxo-prostacyclin, a profound increase in transcript levels was seen for both PDE1C and PDE4B3 together with a slight elevation for PDE4B1. No change in PDE4A transcripts occurred, which was consistent with a lack of change indicated in immunoblotting analyses. In contrast, 7-oxo-prostacyclin treatment caused decrease in transcript levels for PDE4D, which was confirmed by immunoblotting and shown to be due to a reduction in the levels of PDE4D3 and also in PDE4D1/D2. Thus, treatment of animals with 7-oxo-prostacyclin initiated profound isoform-specific changes in PDE expression in the myocardium which, presumably, underpin the increased PDE activity.

Developmental Differences in Distribution of Cyclic Nucleotide Phosphodiesterase Isoforms in Cardiomyocytes and the Ventricular Tissue from Newborn and Adult Rats

We characterized cyclic nucleotide phosphodiesterase (PDE) activities in neonatal rat cardiomyocytes and in whole ventricle from newborn and adult rats, to determine whether cyclic nucleotide hydrolytic activities might be influenced by the developmental stage of the animal or by cell isolation and culture procedures. Using anion-exchange high-performance liquid chromatography (HPLC), we obtained four soluble PDE activities from adult rat ventricle. Peak 1, a cyclic GMP-specific PDE was stimulated by Ca2+/calmodulin; peak 2 hydrolyzed cyclic AMP and cyclic GMP, the addition of cyclic GMP stimulating the hydrolysis of cyclic AMP. Peaks 3 and 4 selectively hydrolyzed cyclic AMP, peak 3 being very sensitive to inhibition by rolipram and peak 4 consisting of two different enzyme activities, one inhibited by cyclic GMP (peak 4b) and the other inhibited by rolipram (peak 4a). In cardiomyocytes and whole ventricle from newborn rats, the response of the two cyclic GMP-sensitive PDE isoforms (cyclic GMP-stimulated and cyclic GMP-inhibited PDE) to the effector cyclic GMP was markedly reduced as compared with that of the corresponding isoforms (peaks 2 and 4b) present in the heart ventricle of adult rats. The other peaks were analogous to peaks 1, 3, and 4a of the whole ventricle. The profile of PDE activities in nonmyocardial cells did not differ from that of myocytes. Therefore, the lack of cyclic GMP effects was not due to culture conditions. Differences in PDE activities observed between cardiomyocytes from newborn rats and ventricle from adult rats might be age dependent.

Differential regulation of the 2′,3′-cyclic nucleotide 3′-phosphodiesterase gene during oligodendrocyte development

The two major isoforms of 2′,3′-cyclic nucleotide phosphodiesterase (CNP), 48 and 46 kDa, have recently been shown to be produced from a single gene by alternative splicing. In addition, messenger RNA encoding the larger isoform is transcribed from a separate promoter, approximately 1 kb upstream from that encoding the smaller isoform. We have investigated the expression of these two CNP isoforms and have found that they are differentially expressed during the process of oligodendrocyte maturation. In oligodendrocyte precursors, only the mRNA encoding the larger protein is found. At the time of oligodendrocyte differentiation, however, both CNP mRNAs are induced. These patterns of CNP expression are likely due to stage-specific transcriptional regulation of the two CNP promoters during the process of oligodendrocyte differentiation.

Isolation and characterization of the rolipram-sensitive cyclic AMP-specific phosphodiesterase (type IV PDE) in human term myometrium

On the basis of the potencies of classical selective modulators of cyclic nucleotide phosphodiesterase (PDE) activities, five cyclic nucleotide PDE isoforms have been isolated and characterized in the cytosolic fraction of human term myometrium. By means of successive ion-exchange chromatographies, a calcium-calmodulin sensitive isoform, a cyclic GMP-stimulated isoform, a cyclic GMP-inhibited isoform, a rolipram-sensitive cyclic AMP-specific isoform and a cyclic GMP-specific isoform, corresponding to PDE I, PDE II, PDE III, PDE IV and PDE V, respectively, have been identified. We found that near term, human myometrium contains a higher proportion of the rolipram-sensitive type IV PDE isoform (about 50% of total cyclic AMP hydrolytic activity) than the type III cyclic GMP-inhibited PDE isoform (only 10%). Type IV PDE displays simple Michaelis-Menten kinetics with a high affinity for cyclic AMP ( K m ∼ 4.4 μM) and is selectively and competitively inhibited by rolipram ( K i ∼ 0.9 μM) and Ro 20-1724 ( K i ∼ 2.6 μM). The predominance of type IV PDE at the end of pregnancy suggests that this isoform contributes, via a modulation of the intracellular cyclic AMP level, to local control of uterine motility and thus could help the myometrium prepare for pronounced contractile activity at the time of parturition.

Concanavalin A stimulates the rolipram-sensitive isoforms of cyclic nucleotide phosphodiesterase in rat thymic lymphocytes

Pretreatment of rat thymic lymphocytes with Concanavalin A induced a very early (30 min) and substantial increase (+90%) of the soluble cAMP phosphodiesterase activity. The crude cytosolic phosphodiesterase activity of rat thymocytes could reproducively be resolved by Mono-Q ion exchange high performance liquid chromatography into four separate phosphodiesterase peaks: a cGMP-stimulated, two cAMP-specific Rolipram-sensitive and a cGMP-inhibited cardiotrope-sensitive peaks. Concanavalin A stimulated very specifically the activity of the two predominant cAMP-specific Rolipram sensitive peaks whereas it only slightly modified the cGMP-stimulated and the cGMP-inhibited forms. The present results strongly suggest that the Rolipram-sensitive cAMP PDE activity may play a key role in the control and regulation of mitogen-induced thymocyte proliferation.

Comparison of cyclic nucleotide phosphodiesterase isoforms from rat heart and bovine aorta: Separation and inhibition by selective reference phosphodiesterase inhibitors

The resolution as well as the biochemical properties of the multiple molecular forms of cyclic nucleotide phosphodiesterase, in a given tissue, may be strongly dependent upon experimental conditions of preparation (extraction of crude enzyme from tissues and fractionation procedures). In the present study, we compare the different molecular forms of cardiac (rat heart ventricle) and vascular (bovine aorta) phosphodiesterase isolated from crude extracts prepared either in sucrose medium or in hypotonic medium (in the presence of protease inhibitors and ion chelators) using two different fractionation procedures: isoelectric focusing on flat gel bed and DEAE-Trisacryl anion exchange chromatography. Both the calmodulin-dependent and the cAMP-specific forms exhibited close IEF and chromatographic patterns and showed similar sensitivities towards reference inhibitors regardless of the tissue of origin. In marked contrast, the cGMP-specific isoform notably differed from one to another tissue with respect to its biochemical properties (only the cardiac tissue being capable of stimulation by cGMP) and sensitivities to xenobiotics. Thus the possibility exists that pharmacological agents may modulate phosphodiesterase activity differently in cardiac and vascular target tissues.

61] High-affinity, calmodulin-dependent isoforms of cyclic nucleotide phosphodiesterase in rat testis

This chapter presents methods to examine changes in the pattern of isoforms during sexual development, and approaches to studying their localization within the various cellular compartments of the testis. To facilitate the latter, in addition to intact testes, methods for studies of Sertoli cell only (SCO) testes, Leydig cell tumors, and purified germ cell suspensions are also presented. The testis, like a wide range of other tissues, contains not one but several forms of cyclic nucleotide phosphodiesterase; three of these appear to be low-k m , calmodulin-dependent isoforms. That these isoforms are not simply artifacts of the chromatographic procedure but distinct isoenzymes is based on differences in substrate specificity, kinetics, thermal stability, and reactions to calmodulin and trifluoperazine. The relatively specific cGMP is prominent in both adult intact and SCO testes, especially in the latter, is the major isoform in the Leydig cell tumor, but is absent in round spermatids. These observations suggest that this phosphodiesterase is more characteristic of the endocrine tissue of the testis rather than the germinal elements, possibly the Leydig cells.

Role of cyclic AMP- and cyclic GMP-phosphodiesterases in the control of cyclic nucleotide levels and smooth muscle tone in rat isolated aorta: A study with selective inhibitors

Three isoforms of cyclic nucleotide phosphodiesterase (PDE) have been recently isolated from aortic tissue and two of them specifically hydrolyzed adenosine 3′,5′-cyclic monophosphate (cAMP) and guanosine 3′:5′-cyclic monophosphate (cGMP), respectively (Lugnier et al., Biochem. Pharmac. 35, 1743, 1986). The role of these forms in controlling cyclic nucleotide levels and smooth muscle tone was investigated by the use of PDE inhibitors. The effects of selective inhibitors of the two forms specifically hydrolyzing cAMP or cGMP (cAMP-PDE and cGMP-PDE, respectively) were compared to those of non-selective inhibitors of the three aortic PDE forms, including the calmodulin-sensitive one (CaM-PDE). Relaxation responses and accumulation of tissue cAMP and cGMP induced by these drugs were studied in precontracted rat isolated aorta, and compared to the effects of isoprenaline and forskolin (stimulants of adenylate cyclase) or sodium nitroprusside (SNP) and sodium azide (stimulants of guanylate cyclase). The eight PDE inhibitors tested all relaxed aorta with potencies that correlated with their potencies as inhibitors of cAMP-PDE, but not of cGMP-PDE. At a concentration producing half-maximal relaxation, all PDE inhibitors induced a moderate but significant accumulation of cAMP, which was comparable to the accumulation of cAMP elicited by half-maximally relaxing concentrations of adenylate cyclase stimulating agents. At this concentration, some PDE inhibitors (M&B 22,948, dipyridamole and to a lesser extent, trequinsin) also induced a significant increase in cGMP levels, of the same order of magnitude as that caused by agents stimulating guanylate cyclase. However, the cGMP-increasing effect of these inhibitors was dissociated from their relaxing effect. In particular, the relaxing concentrations of M&B 22,948 (a selective inhibitor of cGMP-PDE) were clearly higher than the cGMP-increasing concentrations of the compound. At a concentration at which they elicited 10% relaxation by themselves, the selective cAMP-PDE inhibitor, rolipram, as well as the mixed inhibitor of cAMP- and cGMP-PDE, AAL 05 (a cilostamide analogue) enhanced both the cAMP-increasing and the relaxing effect of isoprenaline. Under the same conditions, no clear enhancement of the relaxation induced by SNP was observed. Only M&B 22,948 showed a slight potentiating effect on SNP-induced relaxation, but this effect was limited to low concentrations of SNP (< 10 nM). Conversely, M&B 22,948 inhibited the relaxation elicited by higher concentrations of SNP (⩾ 0.1 μM), and some of the PDE inhibitors completely displaced the concentration-relaxation curve of SNP to the right. One of them, cilostamide, also inhibited the cGMP-increasing effect of SNP, but the other PDE inhibitors did not. The results strongly suggest that inhibition of cAMP-PDE produces relaxation of rat aorta, as a consequence of cAMP accumulation. They do not provide evidence that inhibition of aortic cGMP-PDE can lead by itself to relaxation via cGMP accumulation in this tissue.