Inhibition of cyclic AMP phosphodiesterase activity and myocardial contractility: effects of cilostamide, a novel PDE inhibitor, and methylsiobutylxanthine on rabbit and canine ventricular muscle

The effects of cilostamide (N-cyclohexyl-N-methyl-4-[6-carbostyriloxy]butyramide; OPC-3689), a novel cyclic AMP phosphodiesterase (PDE) inhibitor were compared with those of 1-methyl-3-isobutylxanthine (IBMX) on the rabbit and canine heart preparations. Cilostamide was about three times less potent than IBMX in inhibiting the crude PDE activity of rabbit and canine heart in the cell-free system, while it was 10 times more potent than IBMX in enhancing the positive inotropic action of isoprenaline in the rabbit and canine ventricular myocardium: 10^?6 M cilostamide shifted the concentration-response curve for isoprenaline to the left in a parellel manner to the same extent as did 10^?5 M IBMX. Thus, cilostamide enhanced β-adrenoceptor stimulation more potently than did IBMX and the substances examined previously. Accumulation of intracellular cyclic AMP caused by 10^?6 M isoprenaline in the isolated canine ventricular myocardium was significantly enhanced by 10^?6 M cilostamide and 10^?5 M IBMX; isoprenaline (10^?6 M) induced cyclic AMP accumulation was greater with IBMX (10^?5 M) than with cilostamide (10^?6 M). The threshold concentration for cilostamide itself to induce positive chronotropic and inotropic actions in the rabbit heart was lower than that for IBMX, while the intrinsic activity of IBMX was greater than that of cilostamide. In the canine ventricular myocardium, the positive inotropic actions of cilostamide were comparable to those of IBMX; the action of cilostamide in concentrations of 10^?5 M and higher was partly inhibited by a β-adrenoceptor blocking agent, pindolol (3 × 10^?8 M). During the washout period of the drugs after the maximal response to the drugs had been reached, the positive inotropic action of cilostamide disappeared more rapidly than that of IBMX. The present results suggest that cilostamide is able to permeate the myocardial cell membrane more easily than IBMX and reach the PDE in the functionally important cyclic AMP compartment. The difference in turnover rate of cyclic AMP even in the same tissue in the physiological condition may also affect the direct action of the PDE inhibitors thereon.     

Mechanism underlying the reduced positive inotropic effects of the phosphodiesterase III inhibitors pimobendan,adibendan and saterinone in failing as compared to nonfailing human cardiac muscle preparations

Summary The present study was performed to compare the effects of the new positive inotropic phosphodiesterase III inhibitors pimobendan, adibendan, and saterinone on the isometric force of contraction in electrically driven ventricular trabeculae carneae isolated from explanted failing (end-stage myocardial failure) with those from nonfailing (prospective organ donors) human hearts. In preparations from nonfailing hearts the phosphodiesterase inhibitors, as well as the a-adrenoceptor agonist isoprenaline, the cardiac glycoside dihydroouabain, and calcium, which were studied for comparison, revealed pronounced positive inotropic effects. The maximal effects of pimobendan, adibendan, and saterinone amounted to 56%, 36% and 45%, respectively, of the maximal effect of calcium. In contrast, in preparations from failing hearts the phosphodiesterase III inhibitors failed to significantly increase the force of contraction and the effect of isoprenaline was markedly reduced. The effects of dihydroouabain and calcium were almost unaltered. The diminished effects of isoprenaline were restored by the concomitant application of phosphodiesterase inhibitors.To elucidate the underlying mechanism of the lack of effect of the phosphodiesterase III inhibitors in the failing heart we also investigated the inhibitory effects of these compounds on the activities of the phosphodiesterase isoenzymes I–III separated by DEAE-cellulose chromatography from both kinds of myocardial tissue. Furthermore, the effects of pimobendan and isoprenaline on the content of cyclic adenosine monophosphate (determined by radioimmunoassays) of intact contracting trabeculae were studied. The lack of effect of the phosphodiesterase inhibitors in failing human hearts could not be explained by an altered phosphodiesterase inhibition, since the properties of the phosphodiesterase isoenzymes I–III and also the inhibitory effects of the phosphodiesterase inhibitors on these isoenzymes did not differ between failing and nonfailing human myocardial tissue. Instead, it may be due to a diminished formation of cyclic adenosine monophosphate in failing hearts, presumably caused mainly by a defect in receptor-adenylate cyclase coupling at least in idiopathic dilated cardiomyopathy. Both the basal and the pimobendan-stimulated or isoprenaline-stimulated contents of cyclic adenosine monophosphate of intact contracting trabeculae from failing hearts were decreased compared with the levels in nonfailing hearts. However, under the combined action of isoprenaline and pimobendan the cyclic adenosine monophosphate level reached values as high as with each compound alone in nonfailing preparations, and in addition the positive inotropic effect of isoprenaline was restored.These findings may have important clinical implications. Along with the elevated levels of circulating catecholamines the positive inotropic effects of the phosphodiesterase inhibitors may be maintained in patients with heart failure. Furthermore, the concomitant application of a -adrenoceptor agonist and a phosphodiesterase inhibitor might be beneficial in terminal heart failure refractory to conventional therapeutic regimens.Some of the results reported in this paper have already been presented in abstract form at the 61 st Session of the American Heart Association, Washington, DC, Nov. 1988 (von der Leyen et al., Circulation 78 (Suppl II): 11-360, 1988), at the Fall Meeting of the German Society of Pharmacology and Toxicology, Sept. 1988 (Schmitz et al., Naunyn-Schmiedeberg's Arch Pharmacol 338 (Suppl): R 16, 1988), at the 30th Spring Meeting of the German Society of Pharmacology and Toxicology, March 1989 (Meyer et al., Naunyn-Schmiedeberg's Arch Pharmacol 339 (Suppl): R 53, 1989), and at the XIII Congress of the International Society For Heart Research, Ann Arbor, MI, May 1989 (Meyer et al., J Mol Cell Cardiol 21 (Suppl 11): S. 50, 1989) mis|Send offprint requests to Wilfried Meyer at the above address     

Effects of the phosphodiesterase inhibitor rolipram on the acoustic startle response in rats

Systemic administration of the phosphodiesterase inhibitor rolipram (0.05–10.0 mg/kg, IP) produced a rapid and dose-related increase in the amplitude of the acoustic startle response in rats. The (−) isomer was more potent than the (+) isomer in enhancing startle amplitude. Rolipram increased startle responses that were elicited by brief electrical stimulation of the ventral cochlear nucleus or nucleus reticularis pontis caudalis, two brainstem relay nuclei of the startle neural circuit. A low (5 μg) dose of rolipram produced an excitatory effect on startle following spinal (lumbar intrathecal) infusion but not following supraspinal (lateral ventricle) infusion. Rolipram (0.5 mg/kg, IP) excitation of startle was not blocked by drugs which differentially disrupt the release of monoamines (DSP4, reserpine + alpha-methylpara-tyrosine, reserpine + para-chloro-phenylalanine) or by drugs which differentially block monoamine receptors (haloperidol, prazosin, idazoxan, cinanserin, or cyproheptadine). The marked increase in startle seen following systemic rolipram injection is attributable, at least in part, on an action in the lumbar spinal cord that directly or indirectly facilitates neural transmission along the reticulospinal component of the startle reflex neural pathway. The startle reflex should be a useful behavioral test system for studying the mechanism of action of rolipram and related compounds purported to selectively inhibit calmodulin-independent forms of phosphodiesterase.     

Electrophysiological profile of the antiarrhythmic compound asocainol studied on perfused guinea-pig hearts and on isolated cardiac preparations

Summary The studies deal with electrophysiological effects of asocainol [(±)-6,7,8,9-tetrahydro-2,12-dimethoxy-7-methyl-6-phenetyl-5H-dibenz(d,f)azonine-1-ol] on isolated perfused guinea-pig hearts (Langendorff-preparation), on right ventricular papillary muscles, on Purkinje fibres from the guinea pig, and on isolated sinus nodes from the rabbit.In the perfused heart (n=5) the lowest effective concentration of asocainol is about 0.2 mol/l. At a concentration of 2 mol/l the cardiac electrogram shows in spontaneously beating hearts a mean decrease in frequency of 15%, in electrically driven hearts (150/min at 32°C) prolongation of PQ (+31%), of QRS (+24%) and of QT (+5%).In papillary muscles (32° C; K _e ⁺ 5.9 mmol/l; stimulation rate 0.5 Hz) asocainol (3–30 mol/l) exerts the following effects: no change of the resting potential, concentration-dependent reduction of the maximum rate of rise of the action potential (AP) (–16 to –67%) as well as of the AP-amplitude (–4 to –16%), and shortening of the AP-duration at 50% repolarisation (–18 to –43%). The steady-state dependence of on the resting potential (RP) determined by variation of K _e ⁺ (5.9–15 mmol/l) is shifted by asocainol to more negative potentials. The percentage deviation from controls of the -RP relationship is more pronounced at lower membrane potentials. The influence of asocainol on the recovery from inactivation of shows marked time-dependence. Slow response (Ca²⁺-mediated) APs elicited by strong stimuli in a K _e ⁺ -rich solution (K _e ⁺ 20–24 mmol/l) respond to asocainol (3–10 mol/l) with a marked reduction in amplitude, and duration. In Purkinje fibres the spontaneous activity occurring in K _e ⁺ 2 mmol/l (37°C) is suppressed by asocainol (3–10 mol/l). The excitatory parameters are affected in a way essentially similar to that in papillary muscles. In the primary pacemaker region of the sinus node of the rabbit (37°C) asocainol (10–20 mol/l) reduces the rate of the diastolic depolarisation and diminishes the amplitude and the upstroke velocity of the AP. All the effects of asocainol are only partially reversible by drug-free superfusion for 3 h.The various electrophysiological effects of asocainol are attributed to inhibitory influences of the drug on the fast and on the slow inward current.     

Involvement of cyclic AMP in the direct inotropic action of amrinone

Summary Using the isolated guinea-pig papillary muscle and conventional methods for recording isometric force and transmembrane electrical activity we analysed the positive inotropic effect of the bipyridine derivative amrinone at a contraction frequency of 0.2 Hz. The drug produced a concentration-dependent (0.06–8.0 mmol/l), reversible increase in force of contraction associated with an abbreviation of relaxation time at low concentrations and an increase of this parameter at high concentrations. Part of the inotropic effect was manifested by the rested-state contraction. In muscles depolarized by 24 mmol/l [K]₀, amrinone increased the maximum rate of depolarization and overshoot of the slow responses. Carbachol reduced the inotropic effect of amrinone, and this antagonism was removed by the additional application of atropine. The inotropic effect of amrinone was not affected by propranolol or phentolamine and only slightly inhibited by cimetidine. Amrinone potentiated the positive inotropic effects of isoprenaline or histamine but interacted additively with dihydroouabain; the –log EC₅₀ of isoprenaline was increased by 0.803±0.077 and that of histamine by 1.14±0.054 logarithmic units in the presence of 0.2 mmol/l amrinone. Abbreviation of relaxation time, increase in force of the rested-state contraction, atropine-sensitive antagonism by carbachol, and the effects on the slow response are characteristic of the class of cardiotonic drugs thought to act by increasing the cellular concentration of cyclic AMP. Direct support for this hypothesis was provided by the demonstration that inotropically effective concentrations of amrinone produced an up to 3.5-fold increase in cyclic AMP content of guinea-pig papillary muscles. In addition, amrinone was found to inhibit phosphodiesterase in a crude enzyme preparation from guinea-pig ventricular strips. Lack of specific antagonism by propranolol or cimetidine and potentiation of the effects of isoprenaline or histamine are consistent with an inhibitory effect on cyclic nucleotide phosphodiesterase. At high concentrations, amrinone was shown to exert an additional theophylline-like effect on the contour of the isometric contraction that cannot be attributed to cyclic AMP accumulation.     

The effects of levosimendan and OR-1896 on isolated hearts, myocyte-sized preparations and phosphodiesterase enzymes of the guinea pig

The concentration dependences of the Ca(2+)-sensitizing and the phosphodiesterase-inhibitory effects of levosimendan (the (-) enantiomer of [[4-(1,4,5,6-tetrahydro-4-methyl-6-oxo-3-pyridazinyl)phenyl]hydrazono]propanedinitrile) and its active metabolite, OR-1896 (the (-) enantiomer of N-[4-(1,4,5,6-tetrahydro-4-methyl-6-oxo-3-pyridazinyl)phenyl] acetamide), were compared with their positive inotropic effects to reveal their mechanisms of action in guinea pig hearts. In Langendorff-perfused hearts, left ventricular +dP/dt(max) increased by 26+/-4% and 25+/-3% (mean+/-S.E.M.), with EC(50) values of 15+/-2 and 25+/-1 nM for levosimendan and OR-1896, respectively. In permeabilized myocyte-sized preparations, levosimendan and OR-1896 both increased isometric force production via Ca(2+) sensitization (at pCa 6.2), by 51+/-7% and 52+/-6%, with EC(50) values of 8+/-1 and 36+/-7 nM (P<0.05), respectively. Thus, the two molecules could be defined as Ca(2+) sensitizers and positive inotropes with very similar concentration dependences. However, major differences appeared when the phosphodiesterase-inhibitory effects of levosimendan and OR-1896 were probed on the two phosphodiesterase isoforms (phosphodiesterases III and IV) dominant in the left ventricular cardiac tissue. Levosimendan was a 40-fold more potent and a 3-fold more selective phosphodiesterase III inhibitor (IC(50) for phosphodiesterase III=2.5 nM, and IC(50) for phosphodiesterase IV=25 microM, selectivity factor approximately 10000) than OR-1896 (IC(50) for phosphodiesterase III=94 nM, and IC(50) for phosphodiesterase IV=286 microM, selectivity factor approximately 3000). Hence, our data support the hypothesis that levosimendan and OR-1896 both exert positive inotropy via a Ca(2+)-sensitizing mechanism and not via simultaneous inhibition of the phosphodiesterases III and IV isozymes in the myocardium at their maximal free plasma concentrations.     

Effect of theophylline on calcium exchangeability in ventricular myocardium

Summary The effects of theophylline on contractile force and myocardial calcium exchangeability were studied in isolated, electrically driven Langendorff perfused guinea-pig hearts. Following a 30-min exposure to ⁴⁵Ca, total cellular calcium and ⁴⁵Ca activity were measured in right ventricular samples. Non-toxic theophylline concentrations (5×10^–5–10^–3 g/ml) which augmented contractile force without producing arrhythmias or contractures had no effect on total tissue calcium and did not alter the size of the fraction of cellular calcium exchangeable under steadystate conditions. A toxic concentration of theophylline (2×10^–3 g/ml) induced contractures and increased the amount of exchangeable cellular calcium. The latter effect was due to an increase in total calcium; the unlabelled cellular calcium fraction remained unchanged under the influence of all theophylline concentrations studied. The results suggest that theophylline increases the steady-state calcium exchangeability in ventricular myocardium only when the total calcium concentration is also increased.     

Functional evidence against the existence of β2-adrenoceptors mediating positive inotropic effects in guinea-pig right ventricular myocardium

In guinea-pig isolated papillary muscles we studied the positive inotropic effects of the β-adrenoceptor agonists isoprenaline, fenoterol and noradrenaline in the presence and absence of the β₁-selective antagonist atenolol and the β₂-adrenoceptor antagonist ICI 118.551. In Schild regression analysis pA₂ values for either atenolol (7.14–7.16) and ICI 118.551 (6.79–6.84) were independent of the agonists used. These results support the view that the inotropic response in guinea-pig ventricular myocardium is mediated by β₁-adrenoceptors only. Received: 2 May 1996 / Accepted: 21 August 1996     

Effects of goniopora toxin on bullfrog atrial muscle are frequency-dependent

Summary When goniopora toxin (GPT), a marine toxin isolated from coral, was applied to the bullfrog atrial muscle, the duration of action potential (APD) was prolonged, and a positive inotropic effect was produced. Such effects of GPT were influenced by stimulus frequency. At lower frequencies of 0.1 Hz, GPT (10 to 100 nmol/l) produced a moderate prolongation of APD and positive inotropic effect. At higher prequencies (1.0 Hz), however, the effects of GPT on both APD and contraction were suppressed. In contrast, APD and duration of contraction were prolonged with long intervals of stimulation (1–3 min), in the presence of GPT. The rested-state contraction was also markedly increased and prolonged by GPT. When the membrane potential was conditioned by voltage clamp pulses, the prolonged action potential in GPT-treated muscle was shortened in proportion to the increase in conditioning depolarization. However, the shortening effect of conditioning depolarization was attenuated by lengthening the resting period after the conditioning depolarization. These results, in conjunction with our previous results, suggest that the frequency-dependent effects of GPT on APD and contraction reflect time-and membrane potential-dependent changes of the toxin-modified sodium channels.This work was supported in part by a Grant-in Aid for Special Project Research from the Ministry of Education, Science and Culture of Japan to M. F. (No. 59104007)     

Dissociation of phosphoinositide hydrolysis and positive inotropic effect of histamine mediated by H1-receptors in guinea-pig left atria

Summary The present study was undertaken to determine whether the phosphoinositide hydrolysis is responsible for the positive inotropic effect of histamine in guinea-pig left atria. Histamine induced hydrolysis of phosphoinositides and a positive inotropic effect in a concentration-dependent manner. These effects were antagonized by chlorpheniramine (0.1 mol/l) but not by cimetidine (10 mol/l). At a concentration of 1 mol/l histamine produced a dual-component positive inotropic response composed of an initial increasing phase and a second and late developing, greater positive inotropic phase. Histamine (10 mol/l) caused a gradual increase in the formation of [³H]inositol trisphosphate (IP3) and a significant increase in the [³H]IP₃ level was detected 10 min after the stimulation. Thus, the increase in IP₃ did not precede the increase in force of contraction. The phospholipase C inhibitors 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate (100 mol/l) and neomycin (100 mol/l) significantly reduced the histamine-induced [³H]inositol monophosphate accumulation. However, pretreatment with the phospholipase C inhibitors did not affect the positive inotropic effect of histamine, either in its extent or in its pattern. The phorbol esters 12-O-tetradecanoylphorbol-13-acetate (TPA) (100 nmol/l) and phorbol-12,13-dibutyrate (PDBu) (100 nmol/l) also significantly inhibited the phosphoinositide hydrolysis induced by histamine. The inhibitory effect of the phorbol esters on the phosphoinositide response was completely abolished in the presence of 10 mol/l 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), a protein kinase C inhibitor. TPA significantly attenuated the positive inotropic effect of histamine without changing the dual-component pattern, whereas PDBu merged two distinct components of the histamine inotropic response into one and potentiated the early part of the positive inotropic effect. However, neither of the changes which the phorbol esters produced in the positive inotropic response to histamine was blocked by H-7. In addition, H-7 itself failed to modify the positive inotropic effect of histamine. These results indicate that histamine induces hydrolysis of phosphoinositides in guinea-pig left atria that is mediated by H₁-receptors, but this biochemical event does not appear to contribute to the H₁-receptor-mediated positive inotropic action. Send offprint requests to Y. Hattori at the above address     

Mechanism of erectogenic effect of the selective phosphodiesterase type 5 inhibitor,DA-8159

DA-8159, a new Phosphodiesterase (PDE) 5 inhibitor, has exhibited potent erectogenic potential in a penile erection test in rats and anesthetized dogs. In this study, we investigated the mechanism of its erectogenic activity by measuring the activity of DA-8159 against a various PDE isozymes and assessing cGMP and cAMP formation in a rabbit corpus cavernosum in vitro. DA-8159 inhibited the PDE 5 activity in rabbit and human platelets, which the IC50 was 5.84 +/- 1.70 nM and 8.25 +/- 2.90 nM, respectively. The IC50 of DA-8159 on PDE 1, PDE 2, PDE 3 and PDE 6 were 870+/- 57.4 nM, 101 +/- 15 microM, 52.0 +/- 3.53 microM and 53.3 +/- 2.47 nM, respectively. This suggests that DA-8159 is a potent, highly selective, competitive inhibitor of PDE 5-catalyzed cGMP hydrolysis. The rates of cGMP hydrolysis catalyzed by human platelets-derived PDE 5 as a function of the cGMP concentration (5-100 nM) and two-fixed DA-8159 concentration (11.3 and 18.8 nM) were investigated in order to characterize the mode of PDE 5 inhibition by DA-8159. DA-8159 increased the apparent Km value for cGMP hydrolysis but had no effect on the apparent Vmax, indicating a competitive mode of inhibition. DA-8159 increased the cGMP concentrations in the rabbit corpus cavernosum dose dependently. In the presence of sodium nitroprusside (SNP), DA-8159 significantly stimulated the accumulation of cGMP when compared to the control level. This indicated that the enhancement of a penile erection by DA-8159 involved the relaxation of the cavernosal smooth muscle by NO-stimulated cGMP accumulation. In conclusion, DA-8159 is a selective inhibitor of PDE 5-catalyzed cGMP hydrolysis and the enhancement of a penile erection by DA-8159 is mediated by the relaxation of the cavernosal smooth muscle by the NO-stimulated cGMP accumulation.     

Effects of angiotensin-II on myocardial mechanics and contractile state of heart muscle

The effects of angiotensin-II on the contractile properties of isolated papillary muscle preparations and on the intact dog heart were studied. Angiotensin increased the resting tension, force and velocity of isometric contraction, whilst the time to peak tension and duration of the active state of heart muscle remained unchanged. In isotonic afterloaded conditions, it produced a decrease in the extensibility of the papillary muscles during diastole. The effects of angiotensin on force and velocity of isometric contraction were markedly reduced by β-adrenergic receptor blockade with propranolol or by reserpine pretreatment. Analysis of the afterload-contractile characteristics in intact dog heart revealed a decrease in stroke volume and an increase in left ventricular end-diastolic pressure in the absense of changes in end-diastolic volume.The correlation between the findings of an increased resting tension in isolated heart muscle and those observed in intact hearts indicate that at least part of the inotropic response of heart muscle to angiotensin is caused by the drug directly affecting the final contractile element length.It is concluded that the positive inotropic effect of angiotensin results from two independent actions of the drug on heart muscle. Firstly, it directly increases the resting tension and force of contraction, secondly, through interaction with tyramine-sensitive structures, it indirectly increases the force and velocity of contraction by releasing endogenous catecholamines.     

Erectogenic effect of the selective phosphodiesterase type 5 inhibitor, DA-8159

DA-8159, a new phosphodiesterase 5 inhibitor, was assessed for its erectogenic potential by a penile erection test in rats, the relaxation of isolated rabbit corpus cavernosum (CC), and estimation of the intracavernous pressure (ICP) in the anesthetized dog. Oral administration of DA-8159 (0.3 to 1 mg/kg) increased the number of erections in rats with increasing dosage, with the highest penile erection index at 10 mg/kg. DA-8159 induced the relaxation of phenylephrine (PHE)-induced contractions in the rabbit CC and decreased the IC50 of the nitric oxide donor sodium nitroprusside (SNP) in a dose-dependent fashion. In pentobarbital-anesthetized dogs, the intravenous administration of DA-8159 (1 approximately 300 g/kg) potentiated the increase in ICP induced by the intracavernosal SNP in a dose-related manner. These findings suggest that DA-8159 has significant therapeutic potential in the treatment of erectile dysfunction.     

Effect of a novel inhibitor of cyclic AMP phosphodiesterase,E-1020, on cytosolic Ca++ level and contraction in vascular smooth muscle

Summary 1. The effects of a novel cyclic AMP phosphodiesterase inhibitor, E-1020 (1,2-dihydro-6-methyl-2-oxo5-(imidazo[1,2-a]pyridin-6-yl)-3-pyridine carbonitrile hydrochloride monohydrate), on cytosolic Ca⁺⁺ level ([Ca⁺⁺]_cyt) and muscle tension were examined in rat aorta using a fluorescent Ca⁺⁺ indicator, fura-2. 2. The sustained contraction induced by norepinephrine was more strongly inhibited by E-1020 than that induced by high K⁺. The contraction induced by a higher concentration of the stimulant was less sensitive to E-1020 than that due to a lower concentration. 3. Contractions induced by high K⁺ and norepinephrine followed the increase in [Ca⁺⁺]_cyt. E-1020 inhibited the increments in [Ca⁺⁺]_cyt and muscle tension. A Ca⁺⁺ channel blocker, verapamil, inhibited the norepinephrine-stimulated [Ca⁺⁺]_cyt more strongly than the contraction. E-1020 inhibited the verapamil-insensitive portion of the norepinephrine-stimulated [Ca⁺⁺]_cyt and contraction. 4. Norepinephrine transiently increased [Ca⁺⁺]_cyt and muscle tension in Ca⁺⁺-free solution. E-1020 inhibited the transient contraction but not the stimulated [Ca⁺⁺]_cyt. 5. E-1020 increased the cyclic AMP content of the muscle. The effects of E-1020 on cyclic AMP content and contraction were potentiated by an activator of adenylate cyclase, forskolin. 6. These results suggest that E-1020 inhibits the vascular contractility by the decrease in [Ca⁺⁺]_cyt and decrease in Ca⁺⁺ sensitivity of contractile elements. These effects may be mediated by the increase in cyclic AMP content of the muscle. Send offprint requests to M. Täjimi at the above address     

Actions of the muscle relaxant chandonium iodide on guinea-pig ileum and vas deferens preparations.

The effects of the newly synthesized neuromuscular blocking agent, chandonium iodide (17a-methyl-3beta-pyrrolidino-17a-aza-D-homo-5-androstene dimethiodide) have been investigated on guinea-pig isolated ileum and vas deferens preparations. On the ileum, chandonium (0-1-10-0 mug ml(-1); 1-6 X 10(-7) M-1-6 X 10(-5) M) had weak muscarinic receptor blocking action (pA2 is 5-7), but no antihistamine properties at the concentration tested. No evidence for anticholinesterase actions was found. On the vas deferens, chandonium (10-50 mug ml(-1); 1-6-8-1 X 10(-5) M) potentiated responses to exogenous noradrenaline; responses to electrical stimulation were potentiated only in the presence of 50 mug ml(-1) chandonium. No adrenoceptor or adrenergic neuron blockade was found. The results provide evidence that chandonium acts selectively at acetylcholine receptors and that it is more active as a nicotinic receptor antagonist than as a muscarinic receptor antagonist.     

Effects of adenosine analogues on contractile response and CAMP content in guinea-pig isolated ventricular myocytes

Summary In the present study the effects of adenosine analogues were investigated on cAMP content and contractile response in guinea-pig ventricular myocytes. The adenosine analogues (–)-N⁶-phenylisopropyladenosine (R-PIA), 5-N-ethylcarboxamideadenosine (NECA) and (+)-N⁶-phenyl-isopropyladenosine (S-PIA) in the presence of 0.01 mol/l isoprenaline reduced contractile response concentration-dependently. R-PIA and NECA were about equipotent (IC₂₅: 0.01 mol/l and 0.039 mol/l respectively), while S-PIA was less potent (IC₂₅: 0.6 mol/l). Isoprenaline stimulated cAMP content was reduced by R-PIA (IC₂₅: 0.004 mol/l) and with lower potency by S-PIA (IC₂₅: 0.15 mol/l) but the extent of reduction of cAMP by R-PIA and S-PIA (to 55% and 64% respectively) was less than the reduction of contractile response (to 26% and 55% respectively). This suggests that the effects of R- and S-PIA on contractile response are only in part due to a reduction in cAMP content. In addition, NECA did not decrease cAMP content but decreased contractile response to the same extent as R-PIA. Similar results were obtained in the presence of the phosphodiesterase inhibitor Ro 20-1724. Time course studies revealed that the effects of R-PIA (1 mol/l) on cAMP content and contractile response coincided reaching steady state after 5 min and remained stable thereafter. The effects of NECA (1 µmol/l) on contractility also reached steady state within 5 min, whereas it did not change cAMP content. It is concluded that the reduction of contractility by adenosine analogues in the presence of isoprenaline can only in part be explained by a reduction of cAMP content. It is suggested that a cAMP-independent effect, possibly an activation of phosphatases, might be involved additionally.Abbreviations R-PIA (–)-N⁶-phenylisopropyladenosine - NECA 5-N-ethylcarboxamideadenosine - S-PIA (+)-N⁶-phenylisopropyl-adenosine - Ro 20-1724 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone Send offprint requests to J. Neumann at the above address     

Effects of milrinone on left ventricular end-systolic pressure-volume relationship of rat hearts in situ

1. We have already shown that the left ventricular (LV) end-systolic pressure-volume relationship (ESPVR) of rat hearts in situ is an upward convex curve and that LV end- systolic pressure (ESP(mLVV)) and the systolic pressure-volume area (PVA(mLVV)) at a mid-range LV volume (mLVV) sensitively reflect acute changes in LV contractility and work capability. Milrinone is a non-glycosidic, non-sympathomimetic drug that increases myocardial cAMP concentrations by selective inhibition of cardiac phosphodiesterase III. Therefore, milrinone could act on the entire cardiovascular system and cause an increase in inotropy, arterial vasodilatation and venodilatation. The aim of the present study was to investigate whether the approach we have recently instituted is able to detect the effects of milrinone on the entire cardiovascular system. 2. We measured simultaneously, in anaesthetized rats, continuous LV pressure using a catheter-tip micromanometer and LV volume by LV volumetry using a conductance catheter. We obtained steady state LV pressure-volume loops and intermittently obtained the LV ESPVR by gradual occlusion of the ascending aorta. We then evaluated LV function by assessing milrinone-induced changes in the ESPVR (i.e. ESP(mLVV) and PVA(mLVV)) and vasodilator actions by assessing milrinone-induced changes in ESP(ESV) and effective arterial elastance (Ea), defined as the ESP(ESV)/stroke volume ratio. 3. Milrinone (total dose 49.5 microg; infusion rate 3.3-6.7 microg/min, 7-10 microg/kg per min; blood concentration 53.9 ng/mL) largely shifted the ESPVR upwards and, thus, significantly increased end-systolic pressure (ESP(0.08)) and the systolic pressure-volume area (PVA(0.08)) at a mid-range LV volume (= 0.08 mL/g myocardium). Milrinone also significantly decreased LV ESP(ESV) and decreased Ea, although these decreases were not significant. 4. The results of the present study suggest that our own recently instituted approach to evaluate LV function by measuring LV pressure-volume loops and ESPVR succeeded in detecting a cardiotonic action of milrinone with arterial vasodilatation in normal rat hearts.     

Mechanism of the histamine-induced positive inotropic action in cardiac muscle.

Histamine, a positive inotropic agent which elevates cyclic AMP, was tested for ability to induce Ca2+ channels in 3 preparations of embryonic (16-day-old) chick ventricular myocardial cells whose fast Na+ channels were blocked by tetrodotoxin or voltage inactivated in 25 mM K+. In such inexcitable cells, histamine (10(-6)M) rapidly (1-3 min) induced slowly rising, overshooting, plateau-like responses accompanied by contractions. Mn2+, D600, or H2-receptor blocking agents abolished the slow responses. These results suggest that the positive inotropic action of histamine, like that of catecholamines, is mediated by an increased availability of slow Ca2+ channels.     

Effect of the phosphodiesterase inhibitor 4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone (ZK 62711) on gastric secretion and gastric mucosal cyclic AMP

Summary The effect of the phosphodiesterase inhibitor 4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone (ZK 62711) on gastric secretion and the cyclic AMP system of the gastric mucosa was studied in rats and guinea pigs. In rats, 0.03–0.3 moles/kg ZK 62711 i.v. stimulated acid and pepsin secretion in a dose-dependent manner and 0.03 moles/kg i.v. enhanced the effect of histamine. In guinea pigs no reproducible stimulation was found after intravenous injections of ZK 62711. The stimulation of gastric secretion in the rat by 0.3 moles/kg ZK 62711 i.v. was not affected by vagotomy but was totally inhibited by 10 moles/kg cimetidine i.v. The structurally related phosphodiesterase inhibitor, 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidine (Ro 20-1724), at the dose of 3.3 moles/kg i.v. stimulated gastric secretion in anaesthetised rats to a similar extent as 0.3 moles/kg ZK 62711 i.v. The content of cyclic AMP in the rat gastric mucosa in vivo was slightly increased by 10 moles/kg ZK 62711 i.v, whereas lower doses did not change it. Accumulation of cyclic AMP in the rat gastric mucosa by 50 moles/kg histamine i.v. was enhanced by simultaneous injections of 3.3 moles/kg ZK 62711 i.v. In rat gastric tissue slices in vitro 1–50 M ZK 62711 increased the level of cyclic AMP but 100 M histamine had no effect in the absence or presence of ZK 62711. In gastric mucosal slices of the guinea pig 10 and 50 M ZK 62711 increased the cyclic AMP content which effect was inhibited by 100 M cimetidine and enhanced the stimulatory effect of 100 M histamine on cyclic AMP. The activity of soluble cyclic AMP phosphodiesterase was inhibited by ZK 62711 in the rat (IC₅₀=18 M) and guinea pig gastric mucosa (IC₅₀=1.5 M). Adenylate cyclase was not affected in the homogenate of the guinea pig gastric mucosa. The results indicate that the phosphodiesterase inhibitor ZK 62711 which increases cyclic AMP levels in the gastric mucosa in vivo and in vitro, is a potent stimulator of gastric acid secretion.     

Amelioration of recognition memory impairment associated with iron loading or aging by the type 4-specific phosphodiesterase inhibitor rolipram in rats

Increasing evidence indicates that iron deposition in the brain might play a role in cognitive dysfunction associated with neurodegenerative disorders and aging. Previous studies have not examined whether iron-induced memory deficits can be attenuated by acute treatments with memory-enhancing agents. Phosphodiesterase type 4 (PDE4) inhibitors such as rolipram (ROL) ameliorate memory impairments in several rodent models of amnesia and have been proposed as candidate cognitive-enhancing drugs. Here we show that a single posttraining systemic injection of ROL dose-dependently attenuates the impairment of memory for novel object recognition (NOR) in rats given neonatal iron loading, a model of iron-induced cognitive impairment. Posttraining administration of ROL also recovered NOR deficits associated with aging in rats. These findings provide the first evidence that stimulation of an intracellular second messenger signaling pathway can attenuate iron-induced memory impairment, and support the view that PDE4 inhibitors might ameliorate cognitive dysfunction associated with aging and neurodegenerative disorders.