Mechanisms underlying vasorelaxation induced in the porcine coronary arteries by Thymus linearis, Benth.

ETHNOPHARMACOLOGICAL RELEVANCE: Thymus linearis, Benth indigenous to Pakistan has been traditionally used for the treatment of various diseases including hypertension. AIM OF THE STUDY: Present study aims to investigate vasorelaxant effect of Thymus linearis and its underlying vasorelaxation mechanisms in porcine coronary artery rings. MATERIALS AND METHODS: Aqueous-methanolic extract of aerial parts of Thymus linearis was prepared by maceration process and then bio-guided fractionation was carried out using different solvents. The effects of extract and subsequent fractions were assessed on coronary artery rings with intact and denuded endothelium. The mechanisms of vasorelaxant effect were investigated using different pharmacological tools. The in-vitro inhibitory effects of the test fractions were also assessed on purified phophodiestrases using radioenzymatic assay. Phytochemical studies were carried out using GCMS. RESULTS: The aqueous-methanolic extract elicited similar relaxations in coronary artery rings with and without endothelium in dose dependent fashion and removal of endothelium did not alter this response. Further, n-butanolic fraction of Thymus liniaris (TLB) was found to be the most potent among other derived fractions. TLB did not alter the relaxation produced by endothelium dependent vasodilators in rings with intact endothelium. However, TLB significantly potentiated the relaxation elicited by cyclic AMP and cyclic GMP elevating drugs but not those to soluble guanylyl cyclase activators (YC-1 and BAY 41-2272) and K+ channel openers (levcromakalim and 1-EBIO). Pretreatment with TLB inhibited in a concentration-dependent manner contractions to KCl, CaCl2 and U46619 in coronary artery rings without endothelium. Further, TLB was found to non-selectively inhibit the PDE activity in concentration manner. CONCLUSION: n-Butanolic fraction of Thymus linearis possesses endothelium independent vasorelaxant effects in coronary artery by direct acting on the smooth muscles. These effects involve the elevation of the cyclic AMP and cyclic GMP possibly through the inhibition of various PDEs. GCMS analysis revel presence of thymole and carvacrol as major constituents. Furthermore, these investigations also support the folklore use of Thymus linearis in hypertension.

Endothelium-independent vasorelaxant effect of a Berberis orthobotrys root extract via inhibition of phosphodiesterases in the porcine coronary artery.

BACKGROUND: Berberis orthobotrys Bien ex Aitch. (Berberidaceae) is a plant indigenous of Pakistan that is locally used for the treatment of hypertension. HYPOTHESIS: This study evaluated the vasoactive properties of a Berberis orthobotrys root extract and its fractions, and investigated the role of the endothelium and the underlying mechanism. STUDY DESIGN: An aqueous methanolic extract of Berberis orthobotrys roots was prepared and submitted to a multi-step liquid-liquid fractionation with solvents of increasing polarity. Vascular reactivity of the different fractions was assessed using porcine coronary artery rings either with or without endothelium, and in the presence or absence of specific pharmacological tools. The ability of Berberis orthobotrys extracts to affect phosphodiesterase (PDE) activity was evaluated using a radioenzymatic method and purified phosphodiesterases. RESULTS: The aqueous methanol extract induced similar relaxations in coronary artery rings with and without endothelium, and, amongst the three derived preparations, the butanol fraction (BFBO) was slightly but significantly more effective than the ethyl acetate fraction and the aqueous residue in rings without endothelium. Analysis of the butanol fraction (BFBO) by LC-ELSD-MS indicated the presence of four major isoquinoline alkaloids including berberine. BFBO significantly potentiated the relaxations induced by cyclic GMP- and cyclic AMP-dependent relaxing agonists, and inhibited contractions to KCl, CaCl2, and U46619 in endothelium denuded rings. In contrast, BFBO did not affect relaxations to endothelium-dependent vasodilators. BFBO concentration-dependently inhibited the cyclic GMP-hydrolyzing activity of basal PDE1, calmodulin-activated PDE1 and PDE5, and of cyclic AMP-hydrolyzing activity of PDE3 and PDE4 with IC50 values ranging from 40 to 130µg/ml. CONCLUSION: The butanol fraction of the aqueous methanol extract of Berberis orthobotrys roots induced pronounced endothelium-independent relaxations and inhibited contractile responses by acting directly at the vascular smooth muscle in the coronary artery. Moreover, BFBO potentiated relaxations induced by both cyclic GMP- and cyclic AMP-dependent vasodilators most likely due to its ability to inhibit several vascular PDEs, and in particular PDE4 and PDE5.

Application of drug repositioning strategy to TOFISOPAM.

Drug repositioning strategy is an interesting approach for pharmaceutical companies; especially to increase their productivity. SELNERGY(tm) is a reverse docking based-program able to virtually screen thousands of compounds on more than 2000 3D biological targets. This program was successfully applied to tofisopam and revealed that the isomers of tofisopam are able to fit with phosphodiesterase 4. This old drug was used as a racemic mixture to treat anxiety in the eighties and was recently shown to act as a PDE4 inhibitor. Thanks to this strategy we demonstrated that tofisopam acts via the inhibition of PDE4 in the submicromolar range. Moreover, we firstly showed that the S-enantiomer of tofisopam is ten times more active than R-enantiomer. The identification of the biochemical mechanism of tofisopam isomers now allows to reposition this drug in new therapeutic indications where modulation of cAMP via PDE4 inhibitors are possible.

Short-term or long-term treatments with a phosphodiesterase-4 (PDE4) inhibitor result in opposing agonist-induced Ca(2+) responses in endothelial cells.

BACKGROUND AND PURPOSE: We previously reported that agonist-induced rises in cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) in human umbilical vein endothelial cells (HUVEC) were inhibited after a short-term (2 min) pre-treatment with cAMP-elevating agents. The aim of this work was to study the effects of longer term (8 h) pre-treatment with dibutyryl-cAMP (db-cAMP) or rolipram, a specific inhibitor of phosphodiesterase-4 (PDE4), on [Ca(2+)](i), cAMP levels and PDE activity and expression in HUVEC. EXPERIMENTAL APPROACH: [Ca(2+)](i) changes were measured in isolated HUVEC by Fura-2 imaging. Intracellular cAMP levels and PDE4 activity were assessed by enzyme-immunoassay and radio-enzymatic assay, respectively. PDE expression was measured by northern and western blot analysis. KEY RESULTS: Long-term pre-treatment of HUVEC with rolipram or db-cAMP significantly increased ATP-, histamine- and thrombin-induced [Ca(2+)](i) rises. Short-term pre-treatment with rolipram was associated with an increase in cAMP, whereas long-term pre-treatment was associated with a decrease in cAMP. Long-term pre-treatment with rolipram or db-cAMP induced a significant increase in PDE4 activity and the expression of 74 kDa-PDE4A and 73 kDa-PDE4B was specifically enhanced. All these effects were suppressed by cycloheximide. CONCLUSIONS AND IMPLICATIONS: Our data suggest that sustained inhibition of PDE4 by rolipram induced an increase in PDE4 activity, possibly as a compensatory mechanism to accelerate cAMP degradation and that PDE4A and PDE4B were implicated in the regulation of [Ca(2+)](i). Thus, isozyme-specific PDE4 inhibitors might be useful as therapeutic agents in diseases where [Ca(2+)](i) handling is altered, such as atherosclerosis, hypertension and tolerance to beta-adrenoceptor agonists.

Vascular activity of (-)-anonaine, (-)-roemerine and (-)-pukateine, three natural 6a(R)-1,2-methylenedioxyaporphines with different affinities for alpha1-adrenoceptor subtypes.

We have studied the mechanism of action of three 6a( R)-1,2-methylenedioxyaporphines as vasorelaxant compounds. The alkaloids assayed showed different affinities for the three human cloned alpha (1)-adrenoceptor (AR) subtypes stably expressed in rat-1 fibroblasts, showing lower affinity for alpha(1B)-AR with regard to the alpha(1A)- or alpha(1D)-subtypes. These three natural compounds are more potent inhibitors of [ (3)H]-prazosin binding than of [ (3)H]-diltiazem binding to rat cerebral cortical membranes. As all these alkaloids inhibited noradrenaline (NA)-induced [ (3)H]-inositol phosphate formation in cerebral cortex and rat tail artery, they may be safely viewed as alpha (1)-AR antagonists, as is demonstrated by the vasorelaxant responses observed in isolated rat tail artery and/or aorta precontracted with NA. The alkaloids also inhibited the contractile response evoked by KCl (80 mM) but with a lower potency than that shown against NA-induced contraction. We have also examined their ability to inhibit the different forms of cyclic nucleotide phosphodiesterases (PDE) isolated from bovine aortic smooth muscle and endothelial cells, with negative results. We conclude that N-methylation favours the interaction of (R)-aporphines with all alpha (1)-AR subtypes, and that the topography of the binding site recognizing the basic or protonated nitrogen atom is similar in all three alpha (1)-AR subtypes. The presence of a hydroxy group at C-11 has different effects on the affinity for each alpha (1)-AR subtype but decreases the affinity for Ca (2+) channels. These results confirm and extend the view that subtle changes in the hydroxylation patterns on the aromatic ring of the aporphine structure affect the interactions of these compounds with the three alpha (1)-AR subtypes in different ways, suggesting that the binding site recognizing the aporphine skeleton is different in each of the three subtypes.

Effect of sildenafil on cyclic nucleotide phosphodiesterase activity, vascular tone and calcium signaling in rat pulmonary artery.

(1) Sildenafil (viagra) is a potent PDE5 inhibitor and thus a relaxant drug in corpus carvernosum smooth muscle. In the present work, we evidenced the presence of PDE5 isozyme and investigated the effect of sildenafil on the specific cyclic nucleotide phosphodiesterase (PDE) activity, smooth muscle tone and calcium signaling in the rat main pulmonary artery (MPA). (2) The PDE activity was measured in cytosolic and microsomal fractions. Total cAMP and cGMP-PDE activities were mainly present in the cytosolic fraction. Sildenafil (0.1 micro M) reduced by 72% cGMP-PDE activity, whereas zaprinast (10 micro M), a relatively selective PDE5 inhibitor, reduced this activity by 63%. Sildenafil (0.1 micro M) also inhibited significantly (22%) the cAMP-PDE activity. (3) Western blot analysis revealed the expression of PDE5 mainly in the cytosolic fraction of MPA. Sildenafil concentration-dependently inhibited (IC(50)=3.4 nM) the activity of MPA PDE5 partially purified by HPLC. (4) Sildenafil (0.1 nM-50 micro M) concentration-dependently relaxed MPA rings precontracted with phenylephrine (0.5 micro M). The potency of sildenafil (IC(50)=11 nM) was similar to that of a nitric oxide donor, sodium nitroprusside, but higher than that of zaprinast (IC(50)=600 nM). The vasorelaxant effect of sildenafil was not altered by endothelium removal or in the presence of KT 5823 (1 micro M) and H89 (1 micro M), potent inhibitors of PKG and PKA, respectively. (5) In isolated MPA myocytes, which had been loaded with the calcium fluorophore indo-1, sildenafil (10-100 nM) antagonized ATP- and endothelin-1-induced calcium oscillations but had no effect on the transient caffeine-induced [Ca(2+)](i) response. (6) This study demonstrates the presence of a functional and highly sildenafil-sensitive PDE5 isozyme in rat MPA. Inhibition of this isozyme mainly accounts for the potent pulmonary vasodilator action of sildenafil, which involves alteration in the inositol triphosphate-mediated calcium signaling pathway.

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.

Anti-TNF-alpha properties of new 9-benzyladenine derivatives with selective phosphodiesterase-4- inhibiting properties.

In inflammatory cells, intracellular cAMP concentration is regulated by cyclic nucleotide phosphodiesterases 4. Therefore, PDE4 inhibition appears as a rational goal for treating acute or chronic inflammatory diseases. Selective PDE4 inhibitors have been developed, but due to unwanted side effects, search for new selective PDE4-inhibitors had to be pursued. Recently, Boichot et al. (J. Pharmacol. Exp. Ther. (2000) 292, 647-653) showed that 9-benzyladenine derivatives are selective PDE4 inhibitors. In vivo data in animals suggested that they may induce fewer side effects (emesis). We examined the effects of new 9-benzyladenines on TNF-alpha, interleukin (IL)-1beta, IL-6 and IL-8 production by lipopolysaccharide-activated peripheral blood mononuclear cells, and compared them to other PDEs inhibitors. Selected potent 9-benzyladenines, strongly inhibited TNF-alpha production. Interleukin-1beta, IL-6, and IL-8 production was not significantly affected. Our results suggest that some of these new adenines (i.e., NCS 675 and NCS 700), may be potential therapeutic candidates for the treatment of inflammatory diseases.

Expression and role of phosphodiesterase 6 in the chicken pineal gland.

The chicken pineal gland is directly photosensitive, with light causing an inhibition of melatonin synthesis. A possible role of phosphodiesterase 6 (PDE6, the primary effector of retinal phototransduction) in mediating this response was investigated. RT-PCR, DNA sequencing and northern blots revealed the presence of RNA encoding both catalytic and regulatory subunits of PDE6 in the chicken pineal gland. Both rod and cone forms of PDE6 subunits mRNA were detected. The concentration of the transcripts encoding PDE6 catalytic subunits peaked at night. Western blot analysis of chicken pineal proteins with an antibody directed against the catalytic subunits of bovine rod PDE6 identified a single immunoreactive protein of 97 kDa. Anion exchange chromatography of chicken pineal soluble proteins revealed a peak of PDE6 activity that accounted for about 30% of cyclic GMP-hydrolysis. In cultured chick pineal glands, arylalkylamine N-acetyltransferase (AA-NAT), the rate-limiting enzyme of melatonin synthesis, was protected from inhibition by light when selective PDE5/6 inhibitors (zaprinast, DMPPO) were added to the culture medium. PDE5/6 inhibitors did not affect AA-NAT activity in the dark. In contrast, a general PDE inhibitor (IBMX) increased AA-NAT in a light-independent manner. Together, the data indicate that rod and cone forms of PDE6 are expressed in chick pineal cells and that this enzyme plays a role in the inhibition of melatonin synthesis by light.

Molecular organization of bovine rod cGMP-phosphodiesterase 6.

Phosphodiesterase 6 (PDE6), a multisubunit (alphabetagamma(2)delta) enzyme, plays a major role in visual function by hydrolysing cGMP in response to a light stimulus. Solubilized bovine rod PDE6 molecules depleted of their gamma subunits were purified to homogeneity from bovine retinal rods and their molecular organization was investigated by electron microscopy. Image analysis of single particles revealed the three-dimensional dimeric arrangement of the purified alphabetadelta complex, and the internal organization of each catalytic subunit into three distinct domains at a resolution of 2.8 nm. The relative volume of each domain is consistent with sequence analysis and functional data, which suggest that these domains correspond to the catalytic and two GAF domains. This hypothesis was confirmed by immunolabelling experiments, which located the N-terminal part of the catalytic subunit where the major interaction between the two alphabeta subunits was found to occur. The 3D molecular organization of human platelet PDE5 appears highly homologous to that of bovine rod PDE6, as predicted by similarities in their primary sequences. These observations describe the quaternary organization of the catalytic PDE6 alphabeta complex, and place the catalytic and regulatory domains on a structural model.

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.

Characterization of an in vivo hormonally regulated phosphodiesterase 3 (PDE3) associated with a liver Golgi-endosomal fraction.

The biochemical properties of an in vivo hormonally regulated low Km cAMP phosphodiesterase (PDE) activity associated with a liver Golgi-endosomal (GE) fraction have been characterized. DEAE-Sephacel chromatography of a GE fraction solubilized by a lysosomal extract resulted in the sequential elution of three peaks of activity (numbered I, II, and III), while ion-exchange HPLC resolved five peaks of activity (numbered 1, 2, 3, 4, and 5). Based on the sensitivity of the eluted activity to cGMP and selected phosphodiesterase inhibitors, two phosphodiesterase isoforms were resolved: a cGMP-stimulated and EHNA-inhibited PDE2, eluted in DEAE-Sephacel peak I and HPLC peak 2 and a cGMP-, a cilostamide-, and ICI 118233-inhibited PDE3, eluted in DEAE-Sephacel peak III and HPLC peaks 3, 4, and 5. GE fractions isolated after acute treatments with insulin, tetraiodoglucagon, and growth hormone displayed an increase in phosphodiesterase activity relative to saline-injected controls, as did GE fractions from genetically obese and hyperinsulinemic rats relative to lean littermates. In all experimental rats, an increase in PDE3 activity associated with DEAE-Sephacel peak III and HPLC peaks 4 and 5 was observed relative to control animals. Furthermore, in genetically obese Zucker rats, an increase in the sensitivity of PDE activity to cilostamide and in the amount of PDE activity immunoprecipitated by an antibody to adipose tissue PDE3 was observed relative to lean littermates. These results extend earlier studies on isolated hepatocytes and show that liver PDE3 is the main if not sole PDE isoform activated by insulin, glucagon, and growth hormone in vivo.

Inhibition of type 4 phosphodiesterase by rolipram and Ginkgo biloba extract (EGb 761) decreases agonist-induced rises in internal calcium in human endothelial cells.

The effects of Gingko biloba extract EGb 761 on 5 isolated, vascular, cyclic nucleotide phosphodiesterase (PDE) isoforms were evaluated. EGb 761 preferentially inhibited PDE4 (IC(50)=25.1 mg/L), the isoform that is mainly present in endothelial cells, in a competitive manner (K:(i)=12.5 mg/L). Because changes in cyclic nucleotide levels may affect intracellular calcium ([Ca(2+)](i)) levels in endothelial cells, we examined the effects of EGb 761 on both resting [Ca(2+)](i) levels and agonist-induced rises in [Ca(2+)](i) in single human umbilical vein endothelial cells (HUVECs) in culture. The effects of EGb 761 were compared with those of rolipram, a selective PDE4 inhibitor that increases cellular cAMP levels, and the cAMP analogue dibutyryl cAMP (db-cAMP). EGb 761 (20 and 100 mg/L), rolipram (50 micromol/L), and db-cAMP (100 micromol/L) significantly inhibited histamine-, ATP-, and thrombin-induced [Ca(2+)](i) increases in HUVECs without modifying resting [Ca(2+)](i) levels. Similar results were obtained by using a Ca(2+)-free bath solution. EGb 761 (100 mg/L), but not rolipram (50 micromol/L) or db-cAMP (100 micromol/L), also inhibited Ca(2+) influx into cells having thapsigargin-depleted internal Ca(2+) stores and bathed in a Ca(2+)-free external solution. Our results are consistent with an inhibition of PDE activity that causes a reduction of agonist-induced increases in [Ca(2+)](i) in HUVECs, mainly by inhibition of Ca(2+) mobilization from internal stores. It thus may be that the cardiovascular effects of EGb 761 involve inhibition of PDE4 activity and subsequent modification of Ca(2+) signaling in endothelial cells.

Cyclic nucleotide hydrolysis in bovine aortic endothelial cells in culture: differential regulation in cobblestone and spindle phenotypes.

Cyclic nucleotide phosphodiesterases (PDEs) were investigated in cultured bovine aortic endothelial cells having two phenotypes, cobblestone and spindle, representing, respectively, the resting and angiogenic phenotypes in vivo. Spindle cell homogenates displayed higher hydrolytic activities towards cAMP (52%) and cGMP (10-fold). These increases were due to: (1) increased number of spindle PDE isozymes in the cytosolic fraction (for cAMP: PDE1, PDE2, PDE3 and PDE4 compared to PDE2 and PDE4 in cobblestone; for cGMP: PDE2 and PDE5 compared to PDE2 in cobblestone); (2) increased spindle-specific activities of cytosolic and particulate PDE2, cytosolic PDE3 and particulate PDE4. These changes were associated with an increase in spindle transcripts: 7.5 kb PDE3A (6-fold) and 7.0 kb PDE4D (3-fold). Moreover, cAMP hydrolysis in the two phenotypes was differently regulated by 5 microM cGMP: 60% increase in total cAMP-PDE activity in cobblestone homogenate related to PDE2 stimulation; 30% decrease in spindle homogenate related to PDE3 inhibition. This underlines the roles played by PDE2, PDE3 and PDE5 in the cross-talk involving the two cyclic nucleotides. These changes in PDE isozyme expression along with the cross-talk between cAMP and cGMP may well modulate NO production and consequently might participate in angiogenesis, making PDEs potential targets to modulate angiogenesis.

Coupling of I(1) imidazoline receptors to the cAMP pathway: studies with a highly selective ligand, benazoline.

Clonidine and benazoline are two structurally related imidazolines. Whereas clonidine binds both to alpha(2)-adrenoceptors (alpha(2)R) and to I(1) imidazoline receptors (I(1)R), benazoline showed a high selectivity for imidazoline receptors. Although the alpha(2)R are negatively coupled to adenylate cyclase, no effect on cAMP level by activation of I(1)R has been reported so far. We therefore aimed to compare the effects of clonidine and benazoline on forskolin-stimulated cAMP levels in cell lines expressing either I(1)R only (PC12 cells), alpha(2)R only (HT29 cells), or I(1)R and alpha(2)R together (NG10815 cells). Clonidine proved able to decrease the forskolin-stimulated cAMP level in the cells expressing alpha(2)R and this effect could be blocked by rauwolscine. In contrast, in cells lacking these adrenoceptors, clonidine had no effect. On the other hand, benazoline and other I(1) receptor-selective imidazolines decreased forskolin-stimulated cAMP level in the cells expressing I(1)R, in a rauwolscine- and pertussis toxin-insensitive manner. These effects were antagonized by clonidine. According to these results, we demonstrated that 1) alpha(2)R and I(1)R are definitely different entities because they are expressed independently in different cell lines; 2) alpha(2)R and I(1)R are both implicated in the cAMP pathway in cells (one is sensitive to pertussis toxin and the other is not); and 3) I(1)R might be coupled to more then one transduction pathway. These new data will be essential to further understand the physiological implications of the I(1)R and the functional interactions between I(1) receptors and alpha(2)-adrenoceptors.

Anti-inflammatory activities of a new series of selective phosphodiesterase 4 inhibitors derived from 9-benzyladenine.

Adenine derivatives substituted in position 9 have been demonstrated to have potent phosphodiesterase (PDE) inhibition properties with high selectivity toward PDE4. We compared the effects of various compounds derived from 9-benzyladenine with those of the selective PDE4 inhibitor RP 73401 on the inhibition of PDE4 isolated from bovine aorta, arachidonic acid, and tumor necrosis factor-alpha release by mononuclear cells from healthy subjects. The rank order of potency of the various compounds for in vitro activities on arachidonic acid release is RP 73401 > NCS 613 > NCS 630 > NCS 632 > BWA 78U = NCS 631. The most effective compounds in vitro (RP 73401 and NCS 613) were further investigated in vivo. Both PDE inhibitors dose dependently (1, 10, and 30 mg/kg per os) inhibited the recruitment of neutrophils in the bronchoalveolar lavage fluid of mice exposed to endotoxin via aerosol. Significant differences were observed with 10 and 30 mg/kg RP 73401 and 30 mg/kg NCS 613. In rats, RP 73401, but not NCS 613, significantly increased basal acid secretion at 30 mg/kg i.v. and pentagastrin-stimulated acid secretion at 0.3, 1, and 10 mg/kg. These results demonstrate that the compounds derived from 9-benzyladenine, namely NCS 613, elicit anti-inflammatory activities. It is also suggested that their activities have been mediated through the inhibition of PDE4 isoenzyme. The fact that NCS 613 did not stimulate the gastric acid secretion suggests that this compound may produce fewer gastrointestinal side effects than second-generation PDE4 inhibitors, such as RP 73401.

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.

Effect of pre-exposure to vasoconstrictors on isoprenaline-induced relaxation in rat aorta: involvement of inducible nitric oxide synthase.

1. The aim of this study was to determine whether a brief (30 min) episode of contractile receptor stimulation could affect the degree of a subsequent vasorelaxation. Therefore, concentration - relaxation curves of the rat aorta to isoprenaline were compared before and after exposure of the tissue to noradrenaline (100 microM) or prostaglandin F2alpha (PGF2alpha, 100 microM). 2. Exposure to noradrenaline enhanced the second maximal relaxant effect of isoprenaline (from 20 - 95% relaxation). This effect was not due to significant differences in precontraction levels and was not modified by the presence of the endothelium. Treatment with PGF2alpha mimicked the actions of noradrenaline on subsequent vasorelaxation to isoprenaline. 3. Before exposure to noradrenaline (100 microM), forskolin (10 microM) did not produce any significant relaxation of the rat aorta. After exposure to noradrenaline, forskolin caused a concentration-dependent relaxation with a maximal effect of more than 90% in rings with and without endothelium suggesting that the change in vasorelaxation to isoprenaline occurred downstream from the beta-adrenoceptor. 4. The increase in relaxation due to exposure to noradrenaline was markedly attenuated by treatment with a protein synthase inhibitor (cycloheximide), a nitric oxide (NO) synthase inhibitor (L-NG-nitroarginine methyl ester, L-NAME) and an inhibitor of the activation of soluble guanylyl cyclase (methylene blue). 5. Western blot analysis showed an increase of inducible NO synthase (iNOS) in aortic rings exposed to noradrenaline or PGF2alpha. 6. Together, these findings suggest that pretreatment of rat aorta with noradrenaline or PGF2alpha could induce vascular NOS which would in turn result in an increase in isoprenaline-induced vasorelaxation, this increase occurring downstream from receptor activation. Such a mechanism might participate in cardioprotection during preconditioning induced by noradrenaline.