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Histamine exerts cAMP-dependent positive inotropic effects (PIE) and positive chronotropic effects (PCE) on isolated left and right atria, respectively, of transgenic mice which overexpress the human H2-receptor in the heart (=H2-TG). To determine whether these effects are antagonized by phosphodiesterases (PDEs), contractile studies were done in isolated left and right atrial preparations of H2-TG. The contractile effects of histamine were tested in the additional presence of the PDE-inhibitorserythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride (EHNA, 1 μM, PDE2-inhibitor) or cilostamide (1 μM, PDE3-inhibitor), rolipram (10 μM, a PDE4-inhibitor), and their combinations. Cilostamide (1 μM) and EHNA (1 μM), rolipram (1 μM), and EHNA (1 μM) and the combination of rolipram (0.1 μM) and cilostamide (1 μM) each increased the potency of histamine to elevate the force of contraction (FOC) in H2-TG. Cilostamide (1 μM) and rolipram (10 μM) alone increased and EHNA (1 μM) decreased alone, and their combination increased the potency of histamine to increase the FOC in H2-TG indicating that PDE3 and PDE4 regulate the inotropic effects of histamine in H2-TG. The PDE inhibitors (EHNA, cilostamide, rolipram) alone did not alter the potency of histamine to increase the heart beat in H2-TG whereas a combination of rolipram, cilostamide, and EHNA, or of rolipram and EHNA increased the potency of histamine to act on the beating rate. In summary, the data suggest that the PCE of histamine in H2-TG atrium involves PDE 2 and 4 activities, whereas the PIE of histamine are diminished by activity of PDE 3 and 4.

Serotonin (5-hydroxy-tryptamine, 5-HT) exerted concentration-dependent positive inotropic effects or positive chronotropic effects in transgenic (TG) mice which overexpress the human 5-HT4a receptor in the heart but not in littermate wild-type (WT) mice. These positive inotropic effects and positive chronotropic effects are thought to be mediated by cyclic adenosine 3′,5′-monophosphate (cAMP) in TG cardiomyocytes. To determine whether these effects are antagonized by endogenous phosphodiesterases (PDEs), the inotropic and chronotropic effects of 5-HT were tested in the additional presence of the PDE inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride (EHNA) (1 μM, a PDE2 inhibitor) or cilostamide (1 μM, a PDE3 inhibitor), rolipram (0.1 μM and 1 μM, a PDE4 inhibitor), and their combinations. For comparison, 3-isobutyl-1-methylxanthine (IBMX), an unspecific PDE inhibitor, was investigated. The use of 10 μM IBMX, the combination of rolipram (1 μM) and EHNA (1 μM), and the combination of rolipram (0.1 μM) and cilostamide (1 μM) each increased the potency of 5-HT to elevate the force of contraction in TG mice, but not the potency of 5-HT to increase the beating rate in TG mice. This indicates that PDE4 and PDE2 regulate the inotropic but not the chronotropic effects of 5-HT in TG mice. In contrast, cilostamide (1 μM) alone, EHNA (1 μM) alone, or in combination decreased the potency of 5-HT to increase force of contraction in TG mice. In summary, our present data suggest that the positive chronotropic effect of 5-HT in TG mice does not involve PDE activities, whereas the positive inotropic effect of 5-HT and the basal force in TG mice are diminished by endogenous activity of PDE4. Phosphorylation of PDE4, when PDE2 or PDE3 is inhibited, might enhance the activity of PDE4.

Atrial fibrillation (AF)–associated remodeling includes contractile dysfunction whose reasons are only partially resolved. Serotonin (5-HT) increases contractile force and causes arrhythmias in atrial trabeculae from patients in sinus rhythm (SR). In persistent atrial fibrillation (peAF), the force responses to 5-HT are blunted and arrhythmic effects are abolished. Since force but not arrhythmic responses to 5-HT in peAF could be restored by PDE3 + PDE4 inhibition, we sought to perform real-time measurements of cAMP to understand whether peAF alters PDE3 + PDE4-mediated compartmentation of 5-HT4 receptor-cAMP responses. Isolated human atrial myocytes from patients in SR, with paroxysmal AF (paAF) or peAF, were adenovirally transduced to express the FRET-based cAMP sensor Epac1-camps. Forty-eight hours later, cAMP responses to 5-HT (100 μM) were measured in the absence or concomitant presence of the PDE3 inhibitor cilostamide (0.3 μM) and the PDE4 inhibitor rolipram (1 μM). We successfully established real-time cAMP imaging in AF myocytes. 5-HT increased cAMP in SR, paAF, and peAF, but in line with previous findings on contractility, this increase was considerably smaller in peAF than in SR or paAF. The maximal cAMP response to forskolin (10 μM) was preserved in all groups. The diminished cAMP response to 5-HT in peAF was recovered by preincubation with cilostamide + rolipram. We uncovered a significantly diminished cAMP response to 5-HT4 receptor stimulation which may explain the blunted 5-HT inotropic responses observed in peAF. Since both cAMP and force responses but not arrhythmic responses were recovered after concomitant inhibition of PDE3 + PDE4, they might be regulated in different subcellular microdomains. Electronic supplementary material The online version of this article (10.1007/s00210-020-01968-1) contains supplementary material, which is available to authorized users.

Phosphodiesterases PDE2, PDE3, and PDE4 are expressed in murine sinoatrial cells. PDE3 and/or PDE4 reduce heart rate but apparently do not influence the tachycardia mediated through sinoatrial β1- and β2-adrenoceptors despite the high content of sinoatrial cAMP. The function of PDE2 is, however, uncertain. Prostaglandin PGE1 elicits sinoatrial tachycardia through EP receptors, but the control by phosphodiesterases is unknown. We investigated on spontaneously beating right atria of mice the effects of the PDE2 inhibitors Bay 60-7550 and EHNA on basal beating and the tachycardia produced by noradrenaline (3 nM) and PGE1 (1 μM). Bay 60-7550 (1 μM), but not EHNA (10 μM), increased basal sinoatrial beating. EHNA also failed to produce tachycardia in the presence of the adenosine deaminase inhibitor 2'-deoxycoformycin (10 μM), remaining inconclusive whether PDE2 reduces basal sinoatrial beating. Rolipram (10 μM) and cilostamide (300 nM) caused moderate tachycardia. The tachycardia evoked by Bay 60-7550 was similar in the absence and presence of rolipram. Noradrenaline elicited stable tachycardia that was not increased by Bay 60-7550. A stable tachycardia caused by PGE1 was not increased by the inhibitors of PDE2, PDE3, and PDE4. Unlike PDE3 and PDE4 which reduce murine basal sinoatrial beating, a possible effect of PDE2 needs further research. The stable tachycardia produced by noradrenaline and PGE1, together with the lack potentiation by the inhibitors of PDE2, PDE3, and PDE4, suggests that cAMP generated at the receptor compartments is hardly hydrolyzed by these phophodiesterases. Evidence from human volunteers is consistent with this proposal.

Acting through a low-affinity site of the beta(1)-adrenoceptor (beta(1L)AR), CGP12177 causes sinoatrial tachycardia and positive inotropic effects in left atrium but not in the ventricle of the rat. However, inhibition of either PDE3 or PDE4 also uncovers positive inotropic effects of CGP12177 in ventricle, but whether these phosphodiesterases also control the atrial agonist effects of CGP12177 was unknown. We, therefore, investigated the effects of the PDE3-selective inhibitor cilostamide (300 nM) and PDE4 inhibitor rolipram (1 microM) on the (-)-CGP12177-evoked increases of sinoatrial beating rate and force of paced left atria of the rat. Rolipram (n = 8) increased basal sinoatrial rate by 27 +/- 5 bpm but cilostamide (n = 8) had no effect. The chronotropic potency of (-)-CGP12177 (-logEC(50)M = 7.5) was not changed by rolipram and cilostamide or their combination. (-)-CGP12177 increased left atrial force with intrinsic activity 0.25 compared to (-)-isoprenaline. Rolipram (n = 8) and cilostamide (n = 8) did not change basal force of left atria but concurrent rolipram + cilostamide (n = 8) increased force by 52 +/- 9% of the effect of 200 microM (-)-isoprenaline. Neither rolipram nor cilostamide affected the inotropic potency of (-)-CGP12177 (-logEC(50)M = 7.4) but concurrent rolipram + cilostamide caused potentiation (-logEC(50)M = 8.2) and converted (-)-CGP12177 into a full agonist compared to (-)-isoprenaline. Cyclic AMP appears to maintain sinoatrial rate and PDE4 elicits bradycardia through hydrolysis of cAMP in a compartment distinct from the beta(1L)AR-induced cAMP compartment through which (-)-CGP12177 causes tachycardia. In contrast to the (-)-CGP12177-evoked tachycardia, not controlled by PDE3 and PDE4, these isoenzymes jointly reduce (-)-CGP12177-evoked increases of left atrial contractility through beta(1L)AR.

Activation of both beta(1)- and beta(2)-adrenoceptors increases the contractility of human atrial myocardium through cyclic AMP-dependent pathways. Cyclic AMP is hydrolised by phosphodiesterases, but little is known about which isoenzymes catalyse inotropically relevant cyclic AMP accumulated upon stimulation of beta-adrenoceptor subtypes. We have compared the positive inotropic effects of (-)-noradrenaline and (-)-adrenaline, mediated through beta(1)- and beta(2)-adrenoceptors, respectively, in the absence and presence of the PDE3 inhibitor cilostamide (300 nM) or PDE4 inhibitor rolipram (1 muM) on human atrial trabeculae from non-failing hearts. Cilostamide, but not rolipram, potentiated the effects of both (-)-noradrenaline and (-)-adrenaline. Cilostamide increased the -logEC(50)M of (-)-adrenaline more than of (-)-noradrenaline (P0.05), regardless of whether or not the patients had been chronically treated with beta-blockers. The results are consistent with a greater PDE3-catalysed hydrolysis of inotropically relevant cyclic AMP produced through beta(2)-adrenoceptors than beta(1)-adrenoceptors in human atrium.

Glucagon increases cardiac contractility through G(s) protein-coupled glucagon receptors, but the inotropic responses fade. The fade could be due to receptor desensitisation or to the action of phosphodiesterases (PDE), or to both mechanisms. We investigated the effects of the PDE4 inhibitor rolipram (1 microM) on the inotropic and cAMP-responses to glucagon in paced right ventricular strips of the rat heart. Responses to the partial agonist dobutamine, mediated through beta(1)-adrenoceptors, were studied for comparison. Glucagon increased contractility (-logEC(50)M=7.3 for maximum responses with E(max)=32% of the response to 9 mM Ca(2+)), but the responses tended to fade (-logEC(50)M=7.1 for faded responses with E(max)=11.5%). Dobutamine (-logEC(50)M=5.8, E(max)=56%) produced positive inotropic effects that did not fade. Rolipram did not affect basal contractility and cAMP levels. Rolipram enhanced the contractile responses to glucagon and reduced fade (-logEC(50)M=7.5 and 7.3 with E(max)=74% and 45% for maximum and faded responses respectively). The response to glucagon (0.1 microM) completely faded in the absence of rolipram, but only partially faded and then remained stable in the presence of rolipram (1 microM). Rolipram enhanced contractile responses to dobutamine (-logEC(50)M=6.0, E(max)=75%). Dobutamine (3 microM), but not glucagon (0.1 microM), increased tissue levels of cAMP. Consistent with the inotropic data, rolipram caused glucagon to augment cAMP and enhanced the effects of dobutamine. Thus, PDE4 activity limits the responses mediated through both glucagon receptors and beta(1)-adrenoceptors. PDE4-catalysed hydrolysis of cAMP contributes to the inotropic tachyphylaxis of glucagon.

Inhibitors of phosphodiesterase 4 (PDE4) possess bronchospasmolytic and anti-inflammatory properties, which make them very attractive drugs for the treatment of asthma and COPD. Unfortunately, many PDE4 inhibitors also produce central nervous and gastrointestinal side effects, which have limited their clinical application. PDE4 has two binding sites for the archetypal PDE4 inhibitor rolipram, and it has been suggested that binding to the high-affinity rolipram binding site (HARBS) is responsible for the side effects of PDE4 inhibitors. Recently, we have synthesised the PDE4 inhibitor CC3 which shows low affinity to the HARBS. In the present study we investigated the bronchospasmolytic and anti-inflammatory properties of this novel compound in comparison to rolipram and the PDE3 inhibitor motapizone. The airway-relaxant properties of the PDE inhibitors were analysed in rat precision-cut lung slices (PCLS) in which airways were contracted by methacholine or in passively sensitised PCLS exposed to ovalbumin. The anti-inflammatory properties were investigated by measuring the release of TNF from endotoxin-treated human monocytes.Up to concentrations of 10 microM none of the PDE inhibitors significantly affected bronchoconstriction elicited by 10 microM methacholine. However, if rolipram or CC3 were given in combination with motapizone, methacholine-induced bronchoconstriction was concentration-dependently attenuated. Allergen-induced bronchoconstriction in passively sensitised PCLS was attenuated by CC3 (IC(50) 2.7 microM), rolipram (0.23 microM) and motapizone (8 microM). Combination of equimolar concentrations of motapizone and CC3 (0.34 microM) or rolipram (0.005 microM) showed an additive effect. Endotoxin-induced TNF release from human monocytes was attenuated by all three PDE inhibitors, i.e. CC3 (IC(50) 4.6 microM), rolipram (0.18 microM) and motapizone (5.8 microM). Our findings suggest that PDE4 inhibitors with only low affinity for the HABRS have bronchospasmolytic and anti-inflammatory properties.

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 SKF 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 SKF 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 mediumpIC50 on NA-induced contractions in Ca2+-containing solutionpIC50 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.

The beta-blockers carvedilol and metoprolol provide important therapeutic strategies for heart failure treatment. Therapy with metoprolol facilitates the control by phosphodiesterase PDE3, but not PDE4, of inotropic effects of catecholamines in human failing ventricle. However, it is not known whether carvedilol has the same effect. We investigated whether the PDE3-selective inhibitor cilostamide (0.3 mu M) or PDE4-selective inhibitor rolipram (1 mu M) modified the positive inotropic and lusitropic effects of catecholamines in ventricular myocardium of heart failure patients treated with carvedilol. Right ventricular trabeculae from explanted hearts of nine carvedilol-treated patients with terminal heart failure were paced to contract at 1 Hz. The effects of (-)-noradrenaline, mediated through beta(1)-adrenoceptors (beta(2)-adrenoceptors blocked with ICI118551), and (-)-adrenaline, mediated through beta(2)-adrenoceptors (beta(1)-adrenoceptors blocked with CGP20712A), were assessed in the absence and presence of the PDE inhibitors. The inotropic potency, estimated from -logEC(50)s, was unchanged for (-)-noradrenaline but decreased 16-fold for (-)-adrenaline in carvedilol-treated compared to non-beta-blocker-treated patients, consistent with the previously reported beta(2)-adrenoceptor-selectivity of carvedilol. Cilostamide caused 2- to 3-fold and 10- to 35-fold potentiations of the inotropic and lusitropic effects of (-)-noradrenaline and (-)-adrenaline, respectively, in trabeculae from carvedilol-treated patients. Rolipram did not affect the inotropic and lusitropic potencies of (-)-noradrenaline or (-)-adrenaline. Treatment of heart failure patients with carvedilol induces PDE3 to selectively control the positive inotropic and lusitropic effects mediated through ventricular beta(2)-adrenoceptors compared to beta(1)-adrenoceptors. The beta(2)-adrenoceptor-selectivity of carvedilol may provide protection against beta(2)-adrenoceptor-mediated ventricular overstimulation in PDE3 inhibitor-treated patients. PDE4 does not control beta(1)- and beta(2)-adrenoceptor-mediated inotropic and lusitropic effects in carvedilol-treated patients.

This study was aimed at evaluating the in vitro effects of phosphodiesterase inhibitors and beta2-adrenoceptor agonists on spontaneous contractions of human term myometrium. Rolipram, RP 73401 (3-cyclopentyloxy-N-(3,5(-dichloro-4-pyridil)-4-methoxybenzamide) and Ro 20-1724 (1-4-(3-butoxy-4-methoxybenzyl)-2-imidozolidinone) (phosphodiesterase 4 inhibitors) inhibited spontaneous myometrial contractions (Emax approximately 100%; pD2 of 6.80+/-0.28, 6.84+/-0.32 and 6.31+/-0.03, respectively). Salbutamol and formoterol were less effective (Emax=40+/-6% and 35+/-12%, respectively) than phosphodiesterase 4 inhibitors to reduce myometrial contractility. Inhibitors of phosphodiesterase 3 (milrinone and siguazodan) and 5 (zaprinast) were marginally effective. Rolipram (10-30 nM) and siguazodan (0.1 microM) potentiated the response to salbutamol (Emax=75+/-12%, 88+/-8% and 73+/-12% and pD2=6.51+/-0.20, 6.93+/-0.29 and 6.48+/-0.16, respectively). Sodium nitroprusside (pD2=6.76+/-0.29) and theophylline (pD2=5.15+/-0.22) were effective inhibitors of myometrial contractions. Chromatographic separation of phosphodiesterase isoenzymes demonstrated that phosphodiesterase 4 is predominant but other phosphodiesterase isoenzymes were also identified. In conclusion, phosphodiesterase 4 inhibitors alone or combined with beta2-adrenoceptor agonists have potential interest as tocolytic agents.

The mammalian heart expresses at least five phosphodiesterases (PDE1-5). Catecholamines produce surges of inotropically relevant cAMP through beta(1)-adrenoceptor stimulation. cAMP is mainly hydrolysed by PDE3 and/or PDE4 thereby blunting contractility. Basal sinoatrial beating rate in mouse, rat, piglet and rabbit sinoatrial cells is reduced by PDE3 and/or PDE4 through hydrolysis of cAMP. However, in rodents, the tachycardia elicited by catecholamines through production of cAMP by beta-adrenoceptor activation is not controlled by PDE3 and PDE4, despite a blunting effect of PDE3 or/and PDE4 on basal sinoatrial beating, but it is unknown whether PDE3 limits catecholamine-evoked tachycardia in the rabbit. Since rabbit sinoatrial cells are an important model for pacemaker research, we investigated whether the positive chronotropic effects of (-)-noradrenaline on spontaneously beating right atria of the rabbit are potentiated by inhibition of PDE3 with cilostamide (300 nM). We also studied the sinoatrial effects of the PDE4 inhibitor rolipram (10 microM) and its influence on the responses to (-)-noradrenaline. For comparison, we investigated the influence of cilostamide and rolipram on the positive inotropic responses to (-)-noradrenaline on rabbit left atria and right ventricular papillary muscles. Cilostamide and concurrent cilostamide + rolipram, but not rolipram alone, increased sinoatrial rate by 15% and 31% of the effect of (-)-isoprenaline (200 microM) but the PDE inhibitors did not significantly change the chronotropic potency of (-)-noradrenaline. In contrast in papillary muscle, the positive inotropic effects of (-)-noradrenaline were potentiated 2.4-, 2.6- and 44-fold by cilostamide, rolipram and concurrent cilostamide + rolipram, respectively. In left atrium, the positive inotropic effects of (-)-noradrenaline were marginally potentiated by cilostamide, as well as potentiated 2.7- and 32-fold by rolipram and by concurrent cilostamide and rolipram respectively. To compare the influence of PDE1-5 on basal sinoatrial rate and (-)-noradrenaline-evoked tachycardia, we investigated on rat right atria the effects of selective inhibitors. The PDE4 inhibitor rolipram and non-selective inhibitor isobutyl-methylxanthine caused tachycardia with -logEC(50)s of 7.2 and 5.0 and E(max) of 18% and 102% of (-)-isoprenaline, respectively. Rolipram did not change the chronotropic potency of (-)-noradrenaline. At high concentrations (10-30 microM), the PDE1, PDE3 and PDE5 inhibitors 8-methoxymethyl-3-isobutyl-1-methylxanthine, cilostamide and sildenafil, respectively, caused marginal tachycardia but did not significantly change the chronotropic potency of (-)-noradrenaline. The PDE2-selective inhibitor erythro-9-[2-hydroxy-3-nonyl]adenine caused marginal bradycardia at 30 microM and tended to reduce the chronotropic potency of (-)-noradrenaline. Rabbit PDE3 reduces basal sinoatrial rate. Although PDE4 only marginally reduces rate, under conditions of PDE3 inhibition, it further reduces sinoatrial rate. Both PDE3 and PDE4 control atrial and ventricular positive inotropic effects of (-)-noradrenaline. In contrast, neither PDE3 nor PDE4 limit the sinoatrial tachycardia induced by (-)-noradrenaline. In the rat, only PDE4, but not PDE1, PDE2, PDE3 and PDE5, reduces basal sinoatrial rate. None of the five rat PDEs limits the (-)-noradrenaline-evoked tachycardia. Taken together, these results confirm and expand evidence for our proposal that the cAMP-compartment modulating basal sinoatrial rate, controlled by PDE3 and/or PDE4, is different from the PDE-resistant cAMP compartment involved in beta(1)-adrenoceptor-mediated sinoatrial tachycardia.

Experiments were designed to evaluate the effect of cyclic AMP on the electrically-induced release of noradrenaline from vascular sympathetic nerve terminals. The possible implication of the inhibition of adenylate cyclase in the negative feed-back control by prejunctional alpha 2-adrenoceptors of neurotransmitter release was also investigated. Rat isolated tail arteries were preincubated with [3H]-noradrenaline; the preparations were subsequently perfused/superfused with [3H )-noradrenaline-free medium and their perivascular nerves were field stimulated with 24 pulses at 0.4 Hz (0.3 ms, 200 mA). 2 compounds known to enhance the intracellular concentration of cyclic AMP, namely the membrane permeant analogue 8-Br-cAMP (10-300 mumol/l) and forskolin (0.3-10 mumol/l), an activator of adenylate cyclase, concentration-dependently enhanced the stimulation-evoked tritium overflow. The 1,9-dideoxy derivative of forskolin, which does not stimulate adenylate cyclase, was ineffective. Exposure to the cyclic AMP phosphodiesterase inhibitor rolipram 30 mumol/l produced a moderate increase (about 20%) in tritium overflow. However, in the presence of rolipram the facilitatory effect of forskolin was significantly more pronounced than in its absence. Whereas 8-Br-cAMP produced a slight concentration-dependent enhancement of the stimulation-induced vasoconstriction, forskolin and rolipram depressed it. The alpha 2-adrenoceptor agonist B-HT 933 (3-30 mumol/l) concentration-dependently inhibited the tritium overflow. The effect of B-HT 933 30 mumol/l was slightly, but significantly reduced in the presence of 8-Br-cAMP 100 and 300 mumol/l, but was not changed in the presence of forskolin 3 mumol/l. The facilitatory effect of rauwolscine 1 mumol/l was enhanced in the presence of 8-Br-cAMP 100 mumol/l. During perfusion with 8-Br-cAMP 100 mumol/l, the current strength and frequency were decreased to 150 mA and 0.2 Hz, respectively in order to obtain similar amounts of tritium overflow to those observed in the absence of the cyclic AMP analogue with the initial stimulation parameters. Under these conditions, the inhibition of the overflow by B-HT 933 30 mumol/l and the facilitation by the alpha 2-adrenoceptor antagonist rauwolscine 1 mumol/l were unaltered as compared to controls under initial stimulation conditions. It is concluded that, in the rat tail artery, the terminals of perivascular sympathetic nerves are endowed with an adenylate cyclase system. Cyclic AMP is able to modulate noradrenaline release, but does not appear to play a role in the initiation of the release process itself. In addition, the results do not support the hypothesis that prejunctional alpha 2-adrenoceptors depress noradrenaline release through the inhibition of adenylate cyclase.

The species dependent variation in the cardiotonic activity of selective cyclic nucleotide phosphodiesterase (PDE) isoenzyme inhibitors was examined by comparing the inotropic and PDE inhibitory effects of Org 30029 (N-hydroxy-5,6-dimethoxy-benzo[b]thiophene-2-carboximidamide HCl), 3-isobutyl-1-methyl-xanthine (IBMX), milrinone and rolipram in rat and rabbit ventricular myocardium. The relative activities of PDE isoenzymes in rat and rabbit cardiac ventricle were also examined to assess the role of the different PDE subtypes in modulating contractile force in the two species. In rabbit papillary muscles, IBMX, Org 30029 and milrinone increased contractile force whilst rolipram was inactive. The rank order of potency of the active compounds was Org 30029 greater than IBMX greater than milrinone. Only Org 30029 and IBMX produced significant positive inotropic responses in rat papillary muscles, milrinone and rolipram being inactive. However, large positive inotropic responses were obtained in rat papillary muscles when milrinone and rolipram were tested in combination. In rabbit papillary muscles, the positive inotropic action of milrinone was markedly potentiated by rolipram. Four main types of PDE (I, II, III, IV) isoenzymes were resolved, by DEAE-sepharose or Mono-Q ion-exchange chromatography, from both rat and rabbit cardiac ventricular tissue. In rabbit, Ca2+/calmodulin dependent PDE (PDE I) and cyclic GMP inhibited PDE (PDE III) were the dominant cAMP activities. In contrast, cyclic GMP stimulated PDE (PDE II), PDE III and cGMP insensitive PDE (PDE IV) represented the main cAMP activities in rat cardiac ventricle. The inhibitory effects of Org 30029, IBMX, milrinone and rolipram on PDE isoenzymes from rat and rabbit cardiac ventricle were essentially similar.(ABSTRACT TRUNCATED AT 250 WORDS)

(-)-Noradrenaline and (-)-CGP12177 activate beta(1)-adrenoceptors through a high (H)- and low-affinity (L) site, respectively. The positive inotropic effects of (-)-noradrenaline are blunted by phosphodiesterase4 (PDE4) but not PDE3, while both PDE isoenzymes, acting in concert, prevent the effects of (-)-CGP12177 through beta(1)-adrenoceptors in rat ventricle. We sought to unravel the role of PDE3 and PDE4 on signals through the H and L sites in human myocardium. The kinetics of matching positive inotropic effects of (-)-noradrenaline (20 nM) and (-)-CGP12177 (100 nM) were investigated on human atrial trabeculae in the absence and presence of the PDE3 inhibitor cilostamide (300 nM), PDE4 inhibitor rolipram (1 microM) or both. The influence of cilostamide and rolipram on agonist-evoked cyclic adenosine monophosphate (cAMP) increases were also compared in Chinese hamster ovary (CHO) cells expressing recombinant human beta1 -adrenoceptors. (-)-Noradrenaline and (-)-CGP12177 caused matching inotropic responses that faded during a 60-min time course. Cilostamide, but not rolipram, increased the positive inotropic effects and abolished the time dependent fade of both agonists. In CHO cells, rolipram, but not cilostamide, enhanced the cAMP signals caused by both (-)-noradrenaline and (-)-CGP12177. PDE3, but not PDE4, blunts the positive inotropic effects of both (-)-noradrenaline and (-)-CGP12177 through H and L sites, respectively, of human atrial beta1 -adrenoceptors. However, in CHO cells, PDE4 blunts the cAMP signals of both (-)-noradrenaline and (-)-CGP12177. Neither CHO cells nor the rat ventricle are appropriate models for the beta1 -adrenoceptor-evoked signalling to PDE3 observed in human atrium.

The effect of selective phosphodiesterase (PDE) inhibitors was studied on neural transmission within the enteric nervous system employing a two-compartment bath (containing the oral and the anal end of a segment of guinea-pig ileum, respectively). Ascending excitatory enteric nerve pathways were activated by electrical field stimulation (10 Hz for 2 s, 45 mA, 0.5 pulse duration) in the anal compartment and the resulting contraction of the intestinal circular muscle in the oral compartment was recorded. The partitioned bath enables PDE inhibitors and other drugs to be applied to enteric nerve pathways (in the anal compartment) without interfering with the recording of the smooth muscle contraction in the oral compartment. The PDE 4 inhibitors rolipram (0.01-10 microM) and Ro-20-1724 (0.01-10 microM) significantly (P0.01) inhibited (10-91% and 9-83%, respectively) the nerve-mediated contractions. When both rolipram and Ro-20-1724 were tested after phentolamine (1 microM) or yohimbine (0.1 microM), they were significantly (P0.01) less effective. By contrast prazosin (1 microM) was ineffective. Vinpocetine (50 microM), milrinone (30 microM) and zaprinast (100 microM), which inhibit PDE 1, 3 and 5, respectively, did not modify the nerve-mediated contractions. 8-Bromoadenosine 3',5'-cyclic monophosphate (8-Br-cyclic AMP) or N6,2'-O-dibutyryladenosine 3',5' cyclic monophosphate (dibutyryl cyclic AMP), two analogues of cyclic AMP, at lower concentrations (0.1-1 microM) significantly (P0.01) inhibited (15-73% and 5-49%, respectively) the nerve-mediated contractions, while at higher concentrations (10-100 microM) they caused a significant (P0.01) potentiating (48-68% and 77-78%, respectively) effect. These results indicate that inhibition of PDE 4 (but not PDE 1, PDE 3 or PDE 5) produces a depression of neural transmission within the enteric nervous system, possibly by releasing noradrenaline acting at alpha2-adrenoceptors on enteric neurons.

There is currently interest in the use of inhibitors of cyclic nucleotide phosphodiesterases (PDE) as potential anti-asthma agents. In this study we examined the effects of SCA40 (6-bromo-8-methylaminoimidazol-[1,2-a] pyrazine-2-carbonitrile), a preferential inhibitor of PDE 3 also endowed with PDE 4 and 5 inhibitory activities, on isolated bronchus and eosinophil functions and in an animal model of asthma. SCA40 (1 nM-0.1 mM) produced concentration-dependent inhibition of spontaneous and stimulated tone of human isolated bronchus and reached a maximal relaxation similar to that of theophylline (3 mM). The potency (-log EC50 values) of SCA40 against spontaneous tone (6.52 +/- 0.10) was greater than against tone raised by equieffective concentrations (approximately 70%) of histamine (5.76 +/- 0.06), leukotriene C4 (5.44 +/- 0.11), and acetylcholine (4.98 +/- 0.09). In the presence of cytochalasin B, the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP; 0.5 microM) induced leukotriene C4 production in human eosinophils isolated in discontinuous metrizamide gradients. The production of leukotriene C4 was inhibited by SCA40 in a concentration-related fashion (-log IC50 = 6.04 +/- 0.20; n = 6). Rolipram, a selective PDE 4 inhibitor, was also effective (-log IC50 = 7.29 +/- 0.32) but the selective PDE 3 inhibitor SKF94120 was scarcely effective (10% inhibition for 10 microM). In ovalbumin sensitized guinea-pigs, SCA40 (1 mg kg(-1), i.p.) given 30 min before antigen challenge significantly inhibited the acute bronchoconstriction produced by aerosol antigen (5 mg ml(-1), 30 s) (antigen response was 185 +/- 13 and 91 +/- 21 cmH2O l(-1) s(-1) in control and SCA40-treated animals, respectively, P0.05). Pretreatment with SCA40 (1 mg kg(-1), i.p., 30 min pre- and 3 h post-antigen exposure) prevented airway hyperreactivity to histamine which developed 24 h after exposure of conscious guinea-pigs to aerosol antigen. Eosinophil lung accumulation that accompanied airway hyperreactivity was also inhibited by SCA40 (from 6.15 +/- 0.86 in control to 1.27 +/- 0.27 in treated animals; expressed as eosinophils x 10(6); P0.05). SCA40 (1 mg kg(-1), i.p.) also inhibited the microvascular leakage produced after inhaled antigen (5 mg ml(-1), 30 s) at all airway levels. The haemodynamic effects of SCA40 (1 mg kg(-1), i.p.) consisted of a rapid decrease (peak at 5 min) in mean arterial blood pressure (-39.4 +/- 2.4%) and tracheal mucosal blood flow (-13.5 +/- 2.0%) that slowly recovered with time. These data support previous work showing that PDE inhibition results in antispasmogenic and anti-inflammatory effects. SCA40 was effective in vitro and in vivo and these effects are probably related to its activity as a mixed PDE inhibitor.

There is currently interest in the potential use of selective inhibitors of cyclic nucleotide phosphodiesterases (PDE) in the treatment of asthma. In this study we examined the effects of three selective PDE inhibitors, milrinone (PDE III), rolipram (PDE IV) and zaprinast (PDE V), on the broncoconstriction produced by antigen and histamine, the airway hyperreactivity and microvascular leakage after aerosol exposure to platelet-activating factor (PAF) and antigen, and the antigen-induced eosinophil infiltration in guinea-pig lung. Inhaled rolipram (0.01-10 mg ml-1) inhibited dose dependently the bronchospasm produced by aerosol antigen (5 mg ml-1) an anaesthetised, ventilated guinea-pigs. Rolipram (10 mg ml-1) produced maximal inhibition of antigen-induced bronchoconstriction but only partial inhibition of the response to aerosol histamine (1 mg ml-1). Milrinone and zaprinast (each 10 mg ml-1) showed weak, or no, inhibitory effects against bronchoconstriction produced by aerosol antigen or histamine. Pretreatment with rolipram (10 mg kg-1, i.p.) prevented airway hyperreactivity to histamine which develops 24 h after exposure of conscious guinea-pigs to aerosol PAF (500 micrograms ml-1) or antigen (5 mg ml-1). The pulmonary eosinophil infiltration obtained with 24 h of antigen-exposure was inhibited by rolipram. In contrast, milrinone and zaprinast (each 10 mg kg-1, i.p.) failed to reduce either the airway hyperreactivity of the eosinophil accumulation in these animals. Rolipram (1-10 mg ml-1) reduced the extravasation of Evans blue after aerosol PAF (500 micrograms ml-1) at all airway levels while a lower dose (0.1 mg ml-1) was only effective at intrapulmonary airways. Rolipram (0.01-1 mg ml-1) markedly reduced airway extravasation produced by inhaled antigen (5 mg ml-1). Zaprinast (1-10 mg ml-1) was also effective against airway microvascular leakage produced by aerosol PAF or antigen while milrinone (10 mg ml-1) had no antiexudative effect. These data support previous suggestions that pharmacological inhibition of PDE IV results in anti-spasmogenic and anti-inflammatory effects in the airways and may be useful in the treatment of asthma.

The aim of this study was to use a 3H-adenine pre-labelling technique to characterise the effect of α2-adrenoceptor activation on forskolin-stimulated cyclic AMP accumulation in the isolated porcine palmar lateral vein. Forskolin (10−7–10−4 M) stimulated 3H-cyclic AMP accumulation in the isolated porcine palmar lateral vein in a biphasic and concentration-dependent manner. In the absence of the cyclic AMP-selective phosphodiesterase inhibitor rolipram, forskolin stimulated 3H-cyclic AMP accumulation approximately 7–8 fold. The response reached a peak after 5 min. In the presence of rolipram (10−5 M), basal 3H-cyclic AMP levels were approximately 70% higher than in its absence (basal: 1823 ± 57 dpm; rolipram: 3088 ± 229, n \2 = 3) and forskolin (3 × 10−5 M) stimulated 3H-cyclic AMP accumulation approximately 8 fold. The latter response reached a plateau 10 min after the addition of forskolin. In all subsequent studies, the tissues were incubated with forskolin (3 × 10−5 M) for 5 min in the absence of rolipram. Noradrenaline (NA; 10−9–10−4 M) and UK14304 (10−9−10−4 M) inhibited forskolin-stimulated 3H-cyclic AMP accumulation in a concentration-dependent manner with mean pIC50 values of 7.61 ± 0.37 (n \s = 4) and 7.76 ± 0.23 (n \s = 5), respectively. With either NA or UK14304, the maximal inhibition of the forskolin response obtained was approximately 75%. Neither NA (10−4 M) nor UK14304 (10−4 M) altered basal 3H-cyclic AMP levels. Phenylephrine (10−4 M) had no effect on basal 3H-cyclic AMP levels and produced a 25.4 ± 7.1% inhibition of the forskolin-stimulated response, an effect that was reversed by 10−6 M rauwolscine. Rauwolscine (10−9−10−6 M) produced a concentration-dependent reversal of the inhibitory effect of UK14304 10−6 M on forskolin-stimulated 3H-cyclic AMP accumulation with a mean pKi of 8.35 ± 0.39 (n = 3), but had no effect on basal or on forskolin-stimulated 3H-cyclic AMP levels. Similarly, prazosin (3 × 10−8–3 × 10−5 M) or imiloxan (3 × 10−8––3 × 10−5 M) produced a concentration-dependent reversal of the UK14304 (10−7 M)-induced inhibition of forskolin-stimulated 3H-cyclic AMP accumulation, with mean pKi values of 6.32 ± 0.22 (n = 4) and 6.01 ± 0.30 (n = 3), respectively; neither drug had any effect on basal or on forskolin-stimulated 3H-cyclic AMP levels. This suggests that the receptor is of the α2A-adrenoceptor subtype. It can be seen from these studies that it is possible to measure changes in cyclic AMP accumulation in porcine vascular smooth muscle using a pre-labelling technique, and it has been possible to demonstrate the presence of functional α2-adrenoceptors, stimulation of which results in inhibition of forskolin-stimulated cyclic AMP formation.

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-PDEα), 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-PDEβ), 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-PDEα 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-PDEβ. In a cytosolic fraction from heart ventricle, four distinct PDE isoenzymes, CaM-PDE, cGS-PDE, cGI-PDE and cAMP-PDEβ, were recognized. cAMP-PDEβ 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/PDEα 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-PDEβ, 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-PDEβ, 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 μmol/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.

Chromatographic analysis of 3′,5′-cyclic nucleotide phosphodiesterase (PDE) isoenzymes in the cytosol of human neutrophils shows the predominant presence of PDE IV (cAMP specific) and PDE V (cGMP specific). PDE IV is characterized by (1) cAMP selectivity, (2) a KM for cAMP of 1.2 M and (3) a typical rank order of IC 50-values for PDE inhibitors: 0.13, 0.17, 47 and 9.5 μM for PDE IV selective rolipram, PDE III/IV selective zardaverine, PDE III selective motapizone and unselective 3-isobutyl-l-methylxanthine (IBMX), respectively. Functions of polymorphonuclear leukocytes (PMN) such as N-formylmethionyl-leucyl-phenylalanine (fMLP)-stimulated superoxide release and fMLP/thimerosal elicited leukotriene (LT) biosynthesis are inhibited by these PDE inhibitors with the same rank order and even lower IC50-values. Measurements of changes in cytosolic Cai in Fura-2 loaded PMN demonstrate a transient Cai increase after stimulation with 0.1 μM fMLP and an additional sustained elevation of Cai levels in the presence of thimerosal. PDE inhibitors suppress this sustained phase of Cai release with the same rank order of IC50-values as LT biosynthesis. The correlation between fMLP/thimerosal-induced LT biosynthesis and Cai levels reveal a Cai threshold of 150 nM for arachidonic acid metabolism. cAMP levels in PMN were elevated by PDE inhibitors alone by less than 2-fold. In the presence of fMLP however, cAMP was increased up to 10-fold and the efficacy of PDE inhibitors to increase cAMP paralleled their potency to inhibit PDE IV. It is concluded that (1) suppression of PMN functions is achieved by PDE IV inhibition, (2) necessary cAMP elevations are within 50% increase, (3) superoxide release was affected by cAMP/protein kinase A (PKA) directly whereas (4) for inhibition of LT biosynthesis a cAMP related reduction of Ca-influx is involved.

Downregulation of s-adrenoceptors in response to repeated administration of imipramine or rolipram was investigated in the brain of pregnant rats and ovariectomized animals treated with estrone, progesterone or s1- combination of both hormones. Ovariectomy alone was without influence on drug responses. Pregnancy and complete hormone replacement of animals prior to drug treatment halved the response to imipramine and obliterated that to rolipram. Administration of only estrone to ovariectomized rats did not affect the extent of adrenergic downregulation caused by imipramine, but fully suppressed the rolipram action. Castrated males with or without treatment with estrone and progesterone were used as further controls. Castration alone diminished the effects of both antidepressants. Treatment of gonadectomized rats with both ovarian steroid hormones voided the actions of rolipram. The data indicate that imipramine and rolipram share s1- common pathway for their mechanism of action which can be attenuated by ovarian steroid hormones. In addition, imipramine has s1- second major site of action which is not subject to modulation by female steroids.

Modulation of acetylcholine release via adenosine receptors was studied in rabbit hippocampal slices, which were preincubated with 3H-choline and then continuously superfused. Electrical field stimulation of the slices elicited a release of acetylcholine, which was inhibited in a concentration-dependent manner by various adenosine receptor agonists. The effects of the agonists were antagonized by the methylxanthines. From the order of potency: cyclohexyladenosine greater than (-)phenylisopropyladenosine [-)PIA) greater than 5'-N-ethylcarboxamideadenosine (NECA) greater than 2-chloradenosine greater than (+)phenylisopropyladenosine greater than adenosine, the inhibitory adenosine receptor may be classified as A1-(R1-)receptor. In experiments on rabbit caudate nucleus slices, adenosine receptor agonists only slightly decreased the evoked acetylcholine release. The presence of an inhibitory tone of endogenous adenosine on hippocampal acetylcholine release is supported by the following findings: 1) the methylxanthines theophylline, 8-phenyltheophylline and 3-isobutylmethylxanthine (IBMX) increased the evoked acetylcholine release in concentrations below those required for phosphodiesterase inhibition. 2) Adenosine uptake inhibitors, in contrast, decreased the evoked transmitter release. 3) Deamination of endogenous adenosine by addition of adenosine deaminase to the medium enhanced the acetylcholine release. In conclusion, acetylcholine release in the hippocampus is depressed at the level of the cholinergic nerve terminals by endogenous adenosine via A1-(R1-)receptors.

In reticulocyte-rich suspensions of red blood cells from rats extrusion of cAMP as a regulatory mechanism of intracellular cAMP was investigated. In response to isoprenaline and/or the phosphodiesterase inhibitors Ro 20-1724 and rolipram extrusion of cAMP increases dependent on the concentration of the drugs and time of exposure. However, these drugs exert their effects on the extrusion of cAMP only indirectly, i.e. via increased intracellular levels of cAMP, since the respective EC50-values of the drugs for intracellular accumulation and extrusion of cAMP are identical (isoprenaline: approximately 50 nM; rolipram: approximately 1 microM; Ro 20-1724: 15 microM). The dependence of the rate of extrusion on intracellular levels of cAMP is characterized by a typical concentration-effect relationship from which a maximal capacity of cAMP extrusion of 3-6 nmol/10 min/10(9) cells and a half maximal effective intracellular cAMP concentration of 40-50 nmol/10(9) cells can be derived. This relationship has been inferred from either kinetic or steady-state approaches. At rapidly changing intracellular levels of cAMP an apparent time lag of extracellular cAMP accumulation is obligatorily conditioned by this relationship. Vasodilating drugs which lower the ATP content of the cells as well as the uncoupler of oxidative phosphorylation, FCCP, inhibit the extrusive process (papaverine greater than FCCP greater than dipyridamole greater than dilazep much greater than hexobendine greater than or equal to carbocromene) leading to a 3-5-fold increase of the intrato extracellular concentration gradient of cAMP. It is concluded that extrusion of cAMP is a saturable and energy-dependent process which regulates the intracellular cAMP concentration independent of the activities of adenylate cyclase and phosphodiesterase.

Prolonged pretreatment of rats with the atypical antidepressant rolipram attenuates noradrenaline (NA) sensitivity of the cerebral cortical cAMP generating system. The development of this down-regulation is time (7 d treatment required) and dose dependent (EC50 = 0.35 mg/kg). Density of beta-adrenoceptor as measured by (-)-3H-dihydroalprenolol [(-)-3H-DHA] binding is also reduced by rolipram pretreatment. The effect of rolipram is absolutely stereospecific for the (-)-enantiomer (ED50 = 0.18 mg/kg). In addition, only with this isomer, a reduction in daily weight gain was found compared to sham treated controls. Presynaptic denervation using intracerebroventricular (i.c.v.) injections of 6-hydroxydopamine (6-OHDA) prior to or during rolipram treatment did not completely block the effect of a rolipram treatment on down-regulation of cerebral cortical beta-adrenoceptors. The data favor a pre- and postsynaptic action of rolipram different from all other antidepressants studied so far in this experimental setting. Rolipram is known as inhibitor of brain phosphodiesterase. Using partially purified calmodulin-independent phosphodiesterase from brain it is shown that exclusively the (-)-enantiomer of rolipram inhibits phosphodiesterase with an IC50 of 1.25 mumol/l whereas the (+)-isomer possesses little potency. Since a marked stereospecificity for the (-)-isomer of rolipram was displayed in all pharmacological parameters tested so far with (+)- and (-)-rolipram, it is suggested that stereospecific and isozyme specific inhibition of cAMP-phosphodiesterase is, at least in part, related to the mechanism of action of the potential antidepressant drug rolipram and possibly of other antidepressants as well.

Strips of the rabbit pulmonary artery preincubated with 3H-noradrenaline were superfused with physiological salt solution containing cocaine, corticosterone and propranolol. Basal tritium efflux and electrically evoked tritium everflow were determined. 1. The basal efflux of tritium was not affected by forskolin 0.01–10 μmol/l, 8-Br-cAMP and dibutyryl-cAMP 10–330 μmol/l, or the phosphodiesterase inhibitors rolipram 1–10 μmol/l and AH 21–132 1 μmol/l; it was increased by AH 21–132 10–100 μmol/l. 2. Forskolin concentration-dependently increased the evoked 3H overflow, and this effect was not attenuated by omission of cocaine. The facilitatory effect of forskolin was more pronounced at 0.66 Hz than at 2 Hz. 3. Rolipram, AH 21–132, 8-Br-cAMP or dibutyryl-cAMP also produced a concentration-dependent increase in evoked 3H overflow (8-Br-cAMP was more effective than dibutyryl-cAMP in this respect). Except for the highest concentration investigated, AH 21–132 was more effective in facilitating evoked overflow than in increasing basal efflux. 4. Forskolin, AH 21–132 or 8-Br-cAMP did not alter the percentages of 3H-noradrenaline and 3H-metabolites contained in basal tritium efflux or in stimulation-evoked tritium overflow. 5. When a combination of AH 21–132 plus 8-Br-cAMP or AH 21–132 plus forskolin was administered, the facilitatory effect on evoked tritium overflow was more pronounced than with the single compounds alone. 6. ACTH1–24 also facilitated the evoked tritium overflow. Combined exposure to ACTH1–24 plus forskolin, ACTH1–24 plus AH 21–132 or ACTH1–24 plus forskolin plus AH 21–132 resulted in a clearly more pronounced increase in evoked tritium overflow than exposure to the single compounds alone.

The interactions between putative second effector mechanisms for hydrogen ion secretion were studied in isolated gastric cell preparations of the rat containing 60 - 70% parietal cells. Dibutyryl-CAMP and the compounds which increased the level of cAMP (histamine plus rolipram and forskolin plus rolipram) inhibited the carbachol-induced accumulation of [3H]inositol tris-, bis- and monophosphate. There was both a temporal and quantitative correlation between the increase in cAMP and the inhibition of the accumulation of [3H]inositol phosphates. Cimetidine attenuated the inhibitory effect of histamine on the formation of [3H]inositol phosphates. The enhancement of the accumulation of [3H]inositol phosphates by various concentrations of carbachol affected neither the basal nor the histamine-stimulated cAMP levels. In contrast to dibutyryl-cAMP, dibutyryl-cGMP did not modify the carbachol-induced formation of [3H]inositol phosphates. The biologically active phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), which activates protein kinase C, inhibited both the basal and carbachol-induced accumulation of [3H]inositol phosphates. We suggest that the inhibition of the formation of inositol trisphosphate by the increase in the intracellular level of cAMP and by the activation of protein kinase C might be intracellular negative feedback systems which prevent the overreaction of the acid-secreting parietal cells under the simultaneous influence of the physiological gastric secretagogues.

(1) This study was performed to elucidate the relation between positive inotropy and phosphodiesterase inhibition in the heart. Therefore, the influence on the activity of guinea-pig cardiac phosphodiesterase (PDE) I–III separated by DEAE-cellulose anion exchange chromatography was investigated for the new cardiotonic agents pimobendan, its metabolite UD-CG 212 Cl and milrinone. These effects were compared with those of various other PDE inhibitiors such as IBMX, zaprinast, rolipram and AR-L 57 Cl. A selectivity factor (SF, mean of the IC50 values for PDE I and II inhibition divided by the IC50 for PDE III) was calculated for each drug. The greater this value the more selective was the PDE III inhibition. (2) UD-CG 212 Cl was the most potent (IC50 = 0.19 μmol/l) and most selective inhibitor of PDE III with a SF of 869. Also, selective PDE III inhibitors were pimobendan (SF = 50.5) and milrinone (SF = 70.0) with slightly smaller potencies (IC50 = 2.40 and 1.52 μmol/l, respectively). Zaprinast and rolipram preferentially inhibited PDE I and II, respectively. IBMX and AR-L 57 Cl inhibited PDE I-111 unselectively with similar potencies for all isoenzymes. (3) The PDE inhibitory effects of all substances were compared with their influence on force of contraction (electrically driven papillary muscles) and on frequency of beating (spontaneously beating right auricles) in guinea-pig hearts, thus in preparations of the same species. UD-CG 212 Cl and pimobendan resembled each other in their maximal positive inotropic effects with potencies (EC50) of 1.8 μmol/l and 6.0 μmol/l, respectively. Milrinone exerted a biphasic increase in force of contraction with EC5o values of 0.2 and 695 μmol/l. These substances which selectively inhibited PDE III increased force of contraction maximally by 2.04 mN (UD-CG 212 Cl), 2.77 mN (pimobendan) and 3.29 mN (milrinone). Milrinone increased the frequency of beating by 40%, pimobendan by 22% and UD-CG 212 Cl by 16% at maximal positive inotropic concentrations. (4) The unselective PDE I-III inhibitors IBMX and AR-L 57 Cl increased force of contraction with the highest maximal effects (5.9 mN and 4.23 mN, respectively). There was no correlation between EC5o values for the positive inotropic effects and IC50 values for the inhibition of PDE I–III. Zaprinast exerted only weak (0.87 mN) and rolipram no effects on force of contraction. The latter substances had no positive chronotropic effects. (5) In conclusion, the positive inotropic effects of the new cardiotonic agents pimobendan, UD-CG 212 Cl and milrinone may be at least in part due to selective cardiac PDE III inhibition. For UD-CG 212 Cl and pimobendan the IC50 for PDE III inhibition and the EC50 for the positive inotropic effects were of the same order of magnitude. For milrinone, only the lower EC50 value is similar to the IC50 for the inhibition of PDE III. In the case of the unselective PDE III inhibitors IBMX and AR-1. 57 Cl a factor of 20 was found between potencies for positive inotropy and PDE III inhibition. Thus, PDE III inhibition seems to be a prerequisite for PDE inhibition as a mechanism of action for increasing force of contraction. However, the pronounced positive inotropic effects of IBMX and AR-L 57 Cl provide evidence that a selectivity for PDE III inhibition seems not to be essential for increasing force of contraction. Finally, there is no indication that inhibition of PDE I or II plays a major role in increasing force of contraction.

The adenylate cyclase activator forskolin as well as 8-bromo-cyclic AMP enhanced the electrically evoked release of 3H-noradrenaline and 3H-5-hydroxytryptamine from superfused rat neocortical slices and that of 3H-dopamine from neostriatal slices with comparable EC50's of about 0.5 and 50 microM, respectively, without affecting spontaneous tritium efflux. The phosphodiesterase inhibitor ZK 62771 (3-100 microM) also enhanced 3H-noradrenaline and 3H-dopamine release but slightly reduced 3H-5-hydroxytryptamine release. However, this drug profoundly enhanced spontaneous tritium release in the latter case. The facilitatory effect of forskolin (0.3 microM) on the release of the amine neurotransmitters was potentiated in the presence of ZK 62771 (30 microM). Therefore, cyclic AMP appears to exert a general facilitatory effect on the release of these biogenic amines from central nerve terminals.