Cyclic AMP-mediated regulation of vascular smooth muscle cell cyclic AMP phosphodiesterase activity

1. Rat cultured aortic vascular smooth muscle cells (VSMC) express both cyclic GMP-inhibited cyclic AMP phosphodiesterase (PDE3) and Ro 20-1724-inhibited cyclic AMP phosphodiesterase (PDE4) activities. By utilizing either cilostamide, a PDE3-selective inhibitor, or Ro 20-1724, a PDE4-selective inhibitor, PDE3 and PDE4 activities were shown to account for 15% and 55% of total VSMC cyclic AMP phosphodiesterase (PDE) activity.
2. Treatment of VSMC with either forskolin or 8-bromo-cyclic AMP caused significant concentration- and time-dependent increases in total cellular cyclic AMP PDE activity. Using cilostamide or Ro 20-1724, we demonstrated that both PDE3 and PDE4 activities were increased following forskolin or 8-bromo-cyclic AMP treatment, with a relatively larger effect observed on PDE3 activity. The increase in cyclic AMP PDE activity induced by forskolin or 8-bromo-cyclic AMP was inhibited by actinomycin D or cycloheximide, demonstrating that new mRNA synthesis and protein synthesis were required. An analogue of forskolin which does not activate adenylyl cyclase (1,9-dideoxyforskolin) or an analogue of cyclic GMP (8-bromo-cyclic GMP) did not affect total cyclic AMP PDE activity.
3. Incubation of VSMC with 8-bromo-cyclic AMP for 16 h caused a marked rightward shift in the concentration-response curves for both isoprenaline- and forskolin-mediated activation of adenylyl cyclase. A role for up-regulated cyclic AMP PDE activity in this reduced potency is supported by our observation that cyclic AMP PDE inhibitors (IBMX, cilostamide or Ro 20-1724) partially normalized the effects of isoprenaline or forskolin in treated cells to those in untreated cells.
4. We conclude that VSMC cyclic AMP PDE activity is increased following long-term elevation of cyclic AMP and that increases in PDE3 and PDE4 activities account for more than 70% of this effect. Furthermore, we conclude that increases in cyclic AMP PDE activity contribute to the reduced potency of isoprenaline or forskolin in treated VSMC. These results have implications for long-term use of cyclic AMP PDE inhibitors as therapeutic agents.

     

Negative functional effects of cyclic GMP are altered by cyclic AMP phosphodiesterases in rabbit cardiac myocytes

In this study, we tested the hypothesis that the negative functional effects of cyclic GMP on cardiac myocytes would be affected by the actions of cyclic GMP on cyclic AMP phosphodiesterases. Ventricular myocytes from eight rabbits were used to determine the functional and cyclic AMP changes caused by 10(-7), 10(-6), 10(-5) M 8-Bromo-cGMP alone and after the administration of 10(-6) M milrinone (cyclic GMP-inhibited cyclic AMP phosphodiesterase inhibitor) or 10(-6) M erythro-9-(2-Hydroxy-3-3-nonyl)adenine (EHNA, cyclic GMP-stimulated cyclic AMP phosphodiesterase inhibitor). 8-Br-cGMP dose-dependently reduced %shortening by 35+/-4% of baseline at 10(-5) M. This effect was significantly blunted by EHNA at all doses. The maximum rate of shortening was reduced by 31+/-3% by 10(-5) M 8-Br-cGMP. This effect of 8-Br-cGMP was significantly enhanced (42+/-4%) in the milrinone group. A similar pattern was observed in the maximum rate of relaxation data. Cyclic AMP levels were significantly increased from a baseline level of 4.0+/-0.8 pmol/10(5) myocytes by milrinone (+60%), EHNA (+61%) and 8-Br-cGMP (+47%). The combination of EHNA plus 8-Br-cGMP increased cyclic AMP levels significantly more that the combination of milrinone plus 8-Br-cGMP. Exogenous cyclic GMP reduces myocyte function, while raising cyclic AMP possibly through cyclic GMP-inhibited cyclic AMP phosphodiesterase effects. Blocking cyclic GMP-inhibited cyclic AMP phosphodiesterase enhances the functional effects cyclic GMP, while blocking cyclic GMP-stimulated cyclic AMP phosphodiesterase reduced these effects. The study demonstrated a functional interaction between cyclic GMP and cyclic AMP related to the cyclic GMP affected cyclic AMP phosphodiesterases.     

The effects of theophylline and cyclic AMP on intestinal smooth muscle contractions

The effects of 10^?4-10^?3 M theopphylline, cyclic 3'5'-adenosine monophosphate (cyclic AMP), dibutyryl cyclic AMP (DBC) and theophylline plus DBC were observed on the spontaneous and potassium (K)-induced contractions of the guinea-pig taenia coli. All of the compounds inhibited spontaneous contractions as well as the phasic and tonic components of the K-contracture. The effects of cyclic AMP, a compound which will not readily penetrate the cell membrane, were similar to those of the other compounds in inhibiting the spontaneous and phasic contractions, but it was a less effective inhibitor of the tonic contracture. Inhibition of spontaneous and phasic activity may occur at the cell membrane while the inhibitory actions on the tonic contracture may depend on cellular penetration by the inhibitor. There was no evidence that theophylline and DBC have different sites of action.     

Stimulation of calcium uptake into aortic microsomes by cyclic AMP and cyclic AMP-dependent protein kinase

Summary Enhancement of calcium uptake into rabbit aortic microsomes was seen at a cyclic AMP concentration of 10^–6M in the presence of cyclic AMP-dependent protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37). Protein kinase alone also increased calcium uptake, but cyclic AMP alone was without effect. The results suggest that stimulation of calcium sequestration may be the mechanism of cyclic AMP involvement in vascular smooth muscle relaxation.     

Papaverine, cyclic AMP and the dependence of the rat aorta on extracellular calcium.

The spasmolytic effects of papaverine and dibutyryl cyclic AMP (db-cAMP) were compared on isometric contractile responses induced by addition of increasing amounts of external calcium to K+-depolarized or noradrenaline-stimulated rat aorta strips. Papaverine at a concentration active on depolarized strips (3 times 10(-5) moles/1) reduced the maximal contraction (Emax) elicited by Ca2+ in these preparations, while db-cAMP did not. Contrary to what was observed on depolarized aortae, the degree of inhibition of noradrenaline-stimulated strips did not decrease with increasing extracellular calcium concentration (Ca)e. Both db-cAMP and papaverine at a concentration which did not depress Emax (5 times 10(-6) moles/1) potentiated the relaxing effect of high (Ca)e on contractions elicited by noradrenaline. In conclusion, cyclic AMP is probably implicated in the mode of action of papaverine on the noradrenaline-stimulated rat aorta. At a concentration active on depolarized strips, papaverine is also able to impair contractility directly.     

Activation of PDE2 and PDE5 by specific GAF ligands: delayed activation of PDE5

BACKGROUND AND PURPOSE

By controlling intracellular cyclic nucleotide levels, phosphodiesterases (PDE) serve important functions within various signalling pathways. The PDE2 and PDE5 families are allosterically activated by their substrate cGMP via regulatory so-called GAF domains. Here, we set out to identify synthetic ligands for the GAF domains of PDE2 and PDE5.

EXPERIMENTAL APPROACH

Using fluorophore-tagged, isolated GAF domains of PDE2 and PDE5, promising cGMP analogues were selected. Subsequently, the effects of these analogues on the enzymatic activity of PDE2 and PDE5 were analysed.

KEY RESULTS

The PDE2 ligands identified, 5,6-DM-cBIMP and 5,6-DCl-cBIMP, caused pronounced, up to 40-fold increases of the cAMP- and cGMP-hydrolysing activities of PDE2. The ligand for the GAF domains of PDE5, 8-Br-cGMP, elicited a 20-fold GAF-dependent activation and moreover revealed a time-dependent increase in PDE5 activity that occurred independently of a GAF ligand. Although GAF-dependent PDE5 activation was fast at high ligand concentrations, it was slow at physiologically relevant cGMP concentrations; PDE5 reached its final catalytic rates at 1 µM cGMP after approximately 10 min.

CONCLUSIONS AND IMPLICATIONS

We conclude that the delayed activation of PDE5 is required to shape biphasic, spike-like cGMP signals. Phosphorylation of PDE5 further enhances activity and conserves PDE5 activation, thereby enabling PDE5 to act as a molecular memory balancing cGMP responses to nitric oxide or natriuretic peptide signals.     

Inhibitors of cyclic nucleotide phosphodiesterase 3 and 5 as therapeutic agents in heart failure

Cyclic nucleotide phosphodiesterases (PDE) 3 and 5 regulate cAMP and cGMP signalling in cardiac and smooth muscle myocytes. Important advances in the understanding of the roles of these enzymes have recently been made. PDE3 inhibitors have inotropic and vasodilatory properties, and although they acutely improve haemodynamics in patients with heart failure, they do not improve long-term morbidity and mortality. Although combination therapy with β-adrenergic receptor antagonists or selective inhibition of specific PDE3 isoforms might result in a more favourable long-term outcome, more clinical data are needed to test this proposition. The role of PDE5 inhibitors in the treatment of cardiac disease is evolving. PDE5 inhibitors cause pulmonary and systemic vasodilation. How these drugs will compare with other vasodilators in terms of long-term outcomes in patients with heart failure is unknown. Recent studies also suggest that PDE5 inhibitors may have antihypertropic effects, exerted through increased myocardial cGMP signalling, that could be of additional benefit in patients with heart failure.     

Ocular hypotensive,vasorelaxant and cyclic AMP intermediation activities of clozapine displaying antiglaucoma properties

Clozapine, an atypical antipsychotic agent with multiple receptor antagonist activities, was investigated in vivo and in vitro to discover its effects on intraocular pressure and blood flow, on the contractility of ciliary muscle and nonophthalmologic blood vessels, on calcium concentration in A7r5 smooth muscle cells, and on cyclic AMP intermediation. The adrenergic and muscarinic mechanisms involved in the above effects were also examined. In rabbits, clozapine (0.1, 0.25, and 0.5%) prolonged (IOP) recovery time and inhibited IOP response. Clozapine (0.1 and 0.25%) also produced a significant increase in ocular blood flow in this iris, ciliary, retina, and choroid at 30 to 180 min after drug administration. In isolated guinea pig thoracic aorta, clozapine relaxed phenylephrine (10 μM)- and KCl (75 mM)-induced contractions, the estimated IC₅₀ values being 20.4 ± 3.1 nM and 10.6 ± 1.8 μM, respectively. Clozapine (0.1–100 μM) inhibited phenylephrine (10 μM)-induced influx of Ca²⁺, the estimated IC₅₀ value being 0.4 ± 0.1 nM. In isolated pig eye ciliary muscles, clozapine (1.0–100 μM) inhibited carbachol (10 μM)-induced contractions, the estimated IC₅₀ value being 5.8 ± 1.2 μM. Clozapine (0.1–100 μM) increased cyclic AMP accumulation in pig's ciliary bodies, including ciliary process, ciliary muscle, and trabecular meshwork. Pretreatment with carbachol (100 μM) first decreased, then increased, clozapine-induced cyclic AMP accumulation. Studies of pretreatments with various muscarinic receptor antagonists at 100 μM revealed that pirenzepine significantly enhanced clozapine (100 μM)-induced cyclic AMP accumulations in trabecular meshwork, while 4-DAMP methiodide inhibited it in ciliary bodies, and methoctramine decreased it in ciliary process. The ocular hypotensive effects of clozapine may be mostly related to its muscarinic agonist/antagonist activities and associated cyclic AMP increasing activities, which lead to ciliary muscle relaxation and a possibly associated increase in uveoscleral outflow. Clozapine's ability to increase blood flow and relax vessels may be attributed to its ability to block α-adrenergic and decrease intracellular calcium. IOP is considered the major cause of glaucoma, and compounds which are capable of reducing IOP are considered useful for glaucoma treatment. Based on the results above, clozapine may potentially be important in the development of new antiglaucoma agents. Drug Dev. Res. 44:163–173, 1998. © 1998 Wiley-Liss, Inc.     

Cyclic AMP in the rat jugular vein: relationship to vascular relaxation.

Most blood vessels contract to norepinephrine whereas the rat jugular vein relaxes. In the present study, we sought to determine if differences in the adenylate cyclase system accounted for the differing beta adrenergic responses of blood vessels. Cyclic AMP levels in the rat jugular vein incubated in vitro were lower than levels in the femoral vein or mesenteric artery. In the jugular vein, papaverine, an inhibitor of phosphodiesterase, produced a small, but not significant increase in cyclic AMP levels although in the femoral vein and mesenteric artery, cyclic AMP levels were doubled and tripled, respectively. Norepinephrine increased cyclic AMP only in jugular veins contracted with serotonin or in the presence of papaverine, whereas a marked increase in cyclic AMP occurred in the other vessels in the absence of serotonin or papaverine. In the presence of papaverine, the increase in cyclic AMP by norepinephrine in the jugular vein was less than in the other tissues. Neither the β₁-receptor antagonist, practolol, nor the β₂-receptor antagonist, N-isopropylmethoxamine, blocked the increase in jugular vein cyclic AMP produced by norepinephrine in the presence of papaverine. However, practolol and N-isopropylmethoxamine added together, significantly attenuated the norepinephrine-induced increase in cyclic AMP. These data indicate that relaxation of the rat jugular vein to norepinephrine is not associated with higher resting cyclic AMP levels, or a more responsive β-adrenergic-sensitive adenylate cyclase system.     

Negative chronotropic action of cyclic AMP on the fetal rat heart in organ culture.

Dibutyryl cyclic AMP and cyclic AMP in a DMSO medium were administered to the fetal rat heart in organ culture. Changes in heart rate were observed. Dibutyryl cyclic AMP in concentrations of 5 X 10(-5) M and 1 X 10(-4) M produced significant negative chronotropism. At a concentration of 1 X 10(-3) M, cyclic AMP in a 3% or 0.5% DMSO medium produced significant negative chronotropism. These data were compared with other studies. The negative chronotropic action of cyclic AMP and its dibutyryl derivative are evidence against the involvement of cyclic AMP in positive cardiac chronotropism.     

Effect of papaverine on cyclic nucleotide levels in the isolated rat aorta.

An increase in cAMP but no significant modification in cGMP content could be demonstrated in rat aorta strips after applying papaverine in concentrations which reduced contractile responses. Accumulation of cAMP was induced in noradrenaline-stimulated on K+-depolarized strips, under omission of external Ca2+. Thus the elevation of cAMP level preceded the reduction of contraction subsequently elicited by readdition of Ca2+. The effects could not be dissociated under the experimental conditions used here.     

Relaxation of hormonally stimulated smooth muscular tissues by the 8-bromo derivative of cyclic GMP

Summary Substances that cause contraction or relaxation of smooth muscle have been shown to increase intracellular levels of cyclic GMP. Because of the unclear role of cyclic GMP in the control of smooth muscle tone, cyclic GMP derivatives were exogenously applied to various smooth muscle preparations and their effects on tissue tone were studied.Whereas the basal tone of the rat ductus deferens was not affected by exogenous cyclic GMP or its dibutyryl or 8-bromo derivatives, the contractile responses of this tissue to noradrenaline and acetylcholine were depressed by preincubation with 10 M 8-bromo cyclic GMP (Br-cGMP). The 8-bromo derivatives of 2:3-cyclic GMP, 5-GMP and guanosine were without effects. Cyclic AMP levels were not changed by Br-cGMP. The frequency of oxytocin-stimulated rat uteri was also depressed by Br-cGMP (10 M). In helical strips of rat and rabbit aortae, Br-cGMP (1–100 M) caused a concentration-dependent, rapid decrease in noradrenaline-stimulated tissue tension. Br-2:3-cyclic GMP was ineffective. Noradrenaline-stimulated strips from hog spleen arteries were less sensitive to Br-cGMP than aortic tissue. In ductus deferentes and aortic strips stimulated by K⁺ at a depolarizing concentration, Br-cGMP caused less relaxation than under hormonal stimulation.These findings support the concept that cyclic GMP is involved in the control of smooth muscle tone and that hormone- and drug-induced elevations of the cyclic GMP level can reduce contractile responses to neurotransmitters and hormones.Abbreviations cGMP Guanosine 3:5-monophosphate, cyclic GMP - dibutyryl cGMP N², 2-O-dibutyryl guanosine 3:5-monophosphate - Br-cGMP 8-bromo guanosine 3:5-monophosphate - Br-2:3-cGMP 8-bromo guanosine 2:3-monophosphate - Br-GMP 8-bromo guanosine 5-monophosphate - Br-Guo 8-bromo guanosine, Br-guanosine - cAMP adenosine 3:5-monophosphate, cyclic AMP - dibutyryl cAMP N⁶, 2-O-dibutyryl adenosine 3:5-monophosphate - Br-cAMP 8-bromo adenosine 3:5-monophosphate This work was supported by grants from the Deutsche Forschungsgemeinschaft. Preliminary reports were presented (Schultz, 1977b; Schultz et al., 1978).     

Identification and characterization of isoenzymes of cyclic nucleotide phosphodiesterase in human kidney and heart,and the effects of new cardiotonic agents on these isoenzymes

The present study was done to identify and characterize the isoenzymes of cyclic nucleotide phosphodiesterase (PDE) and to determine their intracellular distribution in human kidney and heart. The in vitro effects of new cardiotonic agents, namely, NSP-805 (4,5-dihydro-5-methyl-6-[4-[(2-methyl-3-oxo-1-cyclopentenyl)amino] phenyl]-3(2H)-pyridazinone), TZC-5665 (6-[4-[2-[3-(5-chloro-2-cyanophenoxy)-2-hydroxypropylamino]-2-methylpropylamino]phenyl]-5-methyl-4,5-dihydro-3(2 H)-pyridazinone) and its metabolites, OPC-18790 ((±)-6-[3-(3,4-dimethoxybenzylamino)-2-hydroxypropoxy]-2-(1H)-quinolinone), MS-857 (4-acetyl-l-methyl-7-(4-pyridyl)-5,6,7,8-tetrahydro-3(2H)-isoquinolinone) and E-1020 (1,2-dihydro-6-methyl-2-oxo-5-(imidazo[1,2-a]pyridin-6yl)-3-pyridine carbonitrile hydrochloride monohydrate), on these human PDE isoenzymes were also investigated.PDE isoenzymes were separated from cytosolic and particulate fractions of homogenates of human kidney and heart by DEAE-Sepharose chromatography. PDE isoenzymes were identified by their elution characteristics, substrate specificities, sensitivities to regulation by effectors and by the use of isoenzyme-specific inhibitors.In a cytosolic fraction from kidney, Ca²⁺/calmodulin-dependent PDE (CaM-PDE), cyclic GMP-stimulated PDE (cGS-PDE), cyclic GMP-inhibited PDE (cGI-PDE) and two forms of cyclic AMP-specific PDE (cAMP-PDE) were resolved. One form of cAMP-PDE (cAMP-PDE), which was eluted at a lower ionic strength than cGI-PDE during DEAE-Sepharose chromatography, was newly recognized in human tissues, though the other form (cAMP-PDE), which eluted later than cGI-PDE, had been previously isolated. Both of these cAMP-specific PDEs had similar properties in that they predominantly hydrolyzed cAMP with similar K _m values for cAMP and were inhibited to almost equal extents by 3-isobutyl-l-methylxanthine (IBMX) but were hardly inhibited by cGMP. However, cAMP-PDE was inhibited about 10 times more weakly (on the basis of IC₅₀ values) by rolipram (4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone) and Ro 20-1724 (4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone than was cAMP-PDE. In a cytosolic fraction from heart ventricle, four distinct PDE isoenzymes, CaM-PDE, cGS-PDE, cGI-PDE and cAMP-PDE, were recognized. cAMP-PDE was the major component of the cAMP-hydrolyzing activity in the cytosolic fraction from human kidney, while CaM-PDE and cGI-PDE represented more than 90% of the total cAMP phosphodiesterase activity in the cytosolic fraction from human heart. In the particulate fractions from human kidney and heart, three activities, those of cGI-PDE and of two forms of cAMP-PDE, were identified. In kidney, cAMP/PDE was the main cAMP-hydrolyzing PDE, while in heart cGI-PDE accounted for most of the activity. Furthermore, we evaluated the inhibitory effects on these human PDE isoenzymes of newly synthesized compounds with inotropic effects, namely, NSP-805, metabolites of TZC-5665 referred to as M-1 (6-(4-aminophenyl)-5-methyl-4,5-dihydro-3(2H)-pyridazinone) and M-2 (6-(4-acetyl-aminophenyl)-5-methyl-4,5-dihydro-3(2H)-pyridazinone), OPC-18790, MS-857 and E-1020. These drugs potently inhibited the activity of cGI-PDE and very weakly inhibited the activities of CaM-PDE and cGS-PDE. With respect to inhibitory effects on cardiac cAMP-PDE, there were some differences between the pyridazinone derivatives, for example NSP-805 and M-2, and the nonpyridazinone derivatives OPC-18790, MS-857 and E-1020. From the IC₅₀ values, it was clear that the pyridazinone derivatives inhibited the activity of cGI-PDE at concentrations that were two to four orders of magnitude lower than that required for the inhibition of the activity of cAMP-PDE, while for the nonpyridazinone derivatives the difference was about one order of magnitude. The inhibition of the activity of human cardiac cGI-PDE by NSP-805, M2, OPC-18790, MS-857 and E-1020 was competitive with respect to cAMP with K _i values of 0.012, 0.32, 0.42, 1.3 and 0.15 mol/l, respectively.These results suggest that there may be two isoforms of cAMP-PDE, which exist not only in the particulate fraction but also in the cytosolic fraction of human tissues, and that PDE inhibitors, which exert their cardiotonic effects by inhibiting cGI-PDE, have different selectivities with respect to the inhibition of the other human PDE isoenzymes. Correspondence to: M. Ito at the above address     

硝普钠与异丙肾上腺素松弛犬气管平滑肌的协同作用

目的：研究硝普钠（ＳＮＰ）与异丙肾上腺素（ＩＳＯ）对犬气管平滑肌松弛作用是否存在协同关系，并分析协同的机制。方法：采用放射免疫分析法测定犬气管平滑肌环核苷酸量，并用磷酸二酯酶（ＰＤＥ）同工酶抑制剂为工具分析ＳＮＰ与ＩＳＯ相互作用的机制。结果：ＳＮＰ（１～１００μｍｏｌ·Ｌ－１）增强ＩＳＯ（３０μｍｏｌ·Ｌ－１）对３μｍｏｌ·Ｌ－１乙酰甲胆碱预收缩的犬气管平滑肌的松弛作用。ＳＮＰ（１００μｍｏｌ·Ｌ－１）与ＩＳＯ（３０μｍｏｌ·Ｌ－１）对犬气管平滑肌松弛的协同作用伴有气管平滑肌中ｃＡＭＰ积聚比ＳＮＰ与ＩＳＯ单独时分别增加１．２倍与０．４倍。ＰＤＥⅤ抑制剂扎普司特（３μｍｏｌ·Ｌ－１）使ＳＮＰ的气管平滑肌ｃＧＭＰ积聚由２．４倍增加到４．６倍，可进一步增加气管松弛作用１．２倍。合并ＰＤＥⅢ抑制剂氰胍哒嗪（３０μｍｏｌ·Ｌ－１）和ＰＤＥⅣ抑制剂咯利普兰（３０μｍｏｌ·Ｌ－１）或合并ＳＮＰ（１００μｍｏｌ·Ｌ－１）和咯利普兰（３０μｍｏｌ·Ｌ－１）均能进一步增强ＩＳＯ的气管ｃＡＭＰ积聚与松弛作用。结论：ＳＮＰ是通过气管平滑肌ｃＧＭＰ含量增加而抑制ＰＤＥⅢ，导致ＳＮＰ与ＩＳＯ对气管松弛的协同作用。     

Vascular smooth muscle cell phosphodiesterase (PDE) 3 and PDE4 activities and levels are regulated by cyclic AMP in vivo

Prolonged incubation of several cell types, including cultured vascular smooth muscle cells (VSMC), with cyclic AMP-elevating agents increases cAMP phosphodiesterase (PDE) activity and levels. In this work, we describe for the first time an increase in arterial VSMC cAMP PDE activity and levels caused by cAMP-elevating agents when these agents are administered to rats in vivo. Injections of rats with dibutyryl cAMP (dbcAMP) or forskolin increased both PDE3 and PDE4 activities in aortic and femoral artery VSMC. Consistent with the idea that cAMP-elevating agents increased PDE3 and PDE4 activities by acting directly on VSMC, local delivery of dbcAMP or forskolin to femoral arteries using a pluronic gel-based approach increased femoral artery VSMC PDE3 and PDE4 activities to levels similar to those observed after injection of these agents. Consistent with a role for de novo mRNA and protein synthesis in the cAMP-elevating agent induced increase in PDE3 and PDE4, 1) systemic administration of forskolin increased PDE3A, PDE3B, and PDE4D mRNA levels in aortic VSMC and femoral artery VSMC, 2) local delivery of dbcAMP increased PDE3A, PDE3B, and PDE4D3 protein levels in femoral artery VSMC, and 3) local administration of either actinomycin D or cycloheximide attenuated the effect of dbcAMP. In addition, our results indicate that the PDE3 and PDE4 variants increased by cAMP-elevating agents in arterial VSMC in situ were distinct from those elevated by these agents in cultured arterial VSMC. Consistent with the effect of increased VSMC cAMP PDE on blood vessel function, inhibition of PDE3 and PDE4 activities potentiated the relaxant effect of forskolin in dbcAMP-treated femoral artery rings to a greater extent than in untreated control blood vessels. We propose that our findings are consistent with the concept that cAMP regulates VSMC cAMP PDE activity and levels in vivo and that VSMC phenotype influences the choice of cAMP PDE variant that is elevated. Our findings are discussed in the context that agents aimed at specific PDE3 or PDE4 variants could perhaps allow greater control of cAMP-mediated regulation of VSMC behaviors that are phenotype-dependent.     

Differential effects of benzamides and thioxanthenes on dopamine-elicited accumulation of cyclic AMP in isolated rabbit retina

Summary Benzamides or thioxanthenes were tested as potential antagonists of the cyclic AMP accumulation induced by 10^–4 M dopamine in intact rabbit retinae in vitro in the presence of 5 to 7 mM theophylline. The neuroleptic sulpiride (10^–4 M) was found to be totally inactive whereas a substituted benzamide (clebopride) had small but significant antagonist effect at the same concentration. Among thioxanthenes, isomers of cisconfiguration, which are potent neuroleptics, completely inhibit the cyclic AMP accumulation induced by dopamine, in contrast to trans-isomers which had no inhibitory effects. These data would confirm that retina in vitro is another suitable model for screening antipsychotic activity of classical neuroleptics and that the mechanism of action of sulpiride still needs further investigation.     

Response of rat small intestinal active aldohexose transport to elevation of mucosal cyclic AMP by forskolin and 3-isobutyl-1-methylxanthine in vitro

Summary The phosphodiesterase-inhibitor 3-isobutyl-1-methylxanthine (IBMX) was able to elevate rat small intestinal cyclic AMP levels to 300% of basal values. Active jejunal d-glucose transport was enhanced parallel to the rise of intracellular cyclic AMP levels to 140% of control values at 100 mol/l IBMX. Transport parameters, as determined in a three compartment model in vitro using a dual label method, indicate increased uphill glucose transport at the site of the brush border membrane, higher intracellular accumulation of the sugar, with unchanged passive permeabilities. Phlorizin-inhibited d-glucose transport and l-glucose transfer in the rat were not affected by the persisting cyclic AMP elevation produced by IBMX. Stimulating effects could also be demonstrated with d-galactose as a substrate. IBMX 100 mol/l also increased active d-glucose as well as 3-O-methylglucose transport in mouse jejunum.Stimulatory effects on intestinal hexose transport and mucosal cyclic AMP levels were also found with the adenylate-cyclase activator forskolin. In the present study, forskolin effects on jejunal mucosal cyclic AMP levels were enhanced in the presence of 100 mol/l IBMX, resulting in a 20-fold increase compared to controls at 20 mol/l forskolin. The concentration response for the effect of forskolin in the presence of 100 mol/l IBMX on d-glucose transport did not produce a significant increase compared to transport stimulation with IBMX alone. At higher concentrations of forskolin however, glucose transport decreased to levels well below the IBMX controls.The elevation of cellular cyclic AMP levels had no effects on passive permeability.Both IBMX 100 mol/l as well as forskolin 20 mol/l inhibited rat jejunal net fluid transport by 40%, combination of both agents resulted in a 55% reduction of net fluid absorption in everted sacs of rat jejunum.These results indicate a functional relationship between jejunal mucosal cyclic AMP levels and active hexose absorption different from the inhibitory role of cyclic AMP in intestinal fluid transport.A preliminary account of this work has been given at the 26th Spring Meeting (March 1985) of the Deutsche Pharmakologische Gesellschaft in Mainz, FRG     

Mechanical Response of Rat Myocardium to Dibutyryl Cyclic AMP in Relation to Effects of α- and β-Adrenoceptor Stimulators

Abstract: Dibutyryl cyclic AMP, and α- and β-adrenoceptor stimulators are all able to elicit inotropic effects, α- and β-Adrenoceptor stimulation are known to change each myocardial contraction-relaxation cycle differently. In order to elucidate the myocardial function of cyclic AMP the effects of dibutyryl cyclic AMP on the contraction-relaxation cycle of isolated rat heart papillary muscle were examined and compared to the effects of α- and β-adrenoceptor stimulation, respectively. Dibutyryl cyclic AMP (in the presence of propranolol) increased developed tension (T_max) by 18%, rate of tension rise (T′_max) by 46%, rate of tension fall (T′_min) by 62% and onset-rate of relaxation (T″_min) by 136%. These changes in the contraction-relaxation cycle were strikingly similar to those produced by isoprenaline (β-adrenoceptor stimulation). The response to dibutyryl cyclic AMP, however, developed much slowlier than did the response to isoprenaline. The latter effect was associated with cyclic AMP elevation in a way indicating a trigger function for cyclic AMP. The α-adrenoceptor stimulation (by phenylephrine combined with propranolol), however, increased measures both for contraction and for relaxation by about the same degree, and the effects occurred without changes of cyclic AMP contents. Phenylephrine alone (combined α- and β-adrenoceptor stimulation) elicited a substantial cyclic AMP elevation but gave mechanical effects only slightly different from the pure α-adrenergic response. Thus cyclic AMP effects did not seem to be fully expressed in this case. As a whole, the results indicate that the effects of both dibutyryl cyclic AMP and of isoprenaline are mediated by the cyclic AMP-system while α-adrenoceptor stimulation involves other mechanisms.     

Activation and induction of cyclic AMP phosphodiesterase (PDE4) in rat pulmonary microvascular endothelial cells

Regulation of the rolipram-sensitive cAMP-specific phosphodiesterase 4 (PDE4) gene family was studied in rat pulmonary microvascular endothelial cells (RPMVECs). Total PDE4 hydrolysis was increased within 10 min after addition of forskolin (10 microM), reached a maximum at 20-40 min, and then gradually declined in the cells. A similar activation of PDE4 activity was observed using a protein kinase A (PKA) activator, N(6)-monobutyryl cAMP. Both the forskolin and the N(6)-monobutyryl cAMP activated PDE4 activities were blocked by the PKA-specific inhibitor, H89. This forskolin-stimulated and PKA-mediated short-term activation of PDE4 activity was further confirmed by in vitro phosphorylation of 87kDa PDE4A6 and 83kDa PDE4B3 polypeptides using exogenous PKA Calpha. Increased immunoreactivity of phosphorylated PDE4A6 in situ was detected in Western blots by a PDE4A-phospho antibody specific to the putative PKA phosphorylation sites. Following long-term treatment of RPMVECs with rolipram and forskolin medium (RFM) for more than 60 days, PDE4 activity reached ten-fold higher values than control RPMVECS with twenty-fold increases detected in intracellular cAMP content. The RFM cells showed increased immunoreactivities of the constitutive 4A6 and 4B3 isoforms plus two novel splice variants at 101kDa (4B1) and 71kDa (4B2). Treatment with H89 did not inhibit the PDE4 elevation in RFM cells. In addition to the increased levels of PDE4 in RFM cells, immunofluorescence showed a translocation of PDE4A and 4B to a nuclear region, which was normally not observed in RPMVECs. The PDE4 activity in RFM cells decayed rapidly with an even faster decline of intracellular cAMP content when forskolin/rolipram were removed from the medium. These results suggest that both the activation (short-term) and induction (long-term) of PDE4A/4B isoforms in RPMVECs are closely modulated by the intracellular cAMP content via both post-translational and synthetic mechanisms.