Antagonism of novel inotropic agents at A1 adenosine receptors and m-cholinoceptors in human myocardium

Summary The effects of the new inotropic agents saterinone, sulmazole, UD-CG 212.C1 and milrinone at A₁ adenosine receptors and m-cholinoceptors were evaluated in human myocardium from patients with heart failure. At A₁ adenosine receptors, all compounds inhibited ³H-DPCPX-binding to ventricular membrane preparations at micromolar concentrations. As judged from the K_i-values, the rank order of potency was saterinone > sulmazole > UD-CG 212.C1 > milrinone. The new inotropic agents also displaced the binding of ³H-QNB at m-cholinoceptors. Except for saterinone, the concentration ranges of mean K_i-values were considerably higher at m-cholinoceptors than at A₁ adenosine receptors. The rank order of potency was saterinone > sulmazole > UD-CG 212.Cl > milrinone. Competition of the A₁ adenosine receptor agonist R-PIA to ³H-DPCPX-binding showed a biphasic curve with a shallow slope (Hill coefficient n_H = 0.63) and revealed two affinity states of the A₁ adenosine receptor. In the presence of guanine nucleotides [Gpp(NH)p], the competition curve showed one low affinity class of binding sites and was shifted to the right. In contrast, the competition curves of the new inotropic agents were characterized by a monophasic, steeper slope (mean Hill coefficient n_H = 0.98). Guanine nucleotides had no effect. Similar results were obtained with saterinone and carbachol at m-cholinoceptors. Competition with carbachol revealed three affinity states of the m-cholinoceptor, the superhigh affinity binding was reversed by Gpp(NH)p. Competition with saterinone revealed one class of binding sites which was not influenced by Gpp(NH)p. Accordingly, in isolated, electrically driven human atrial trabeculae, the negative inotropic effect of adenosine was antagonized concentration-dependently by saterinone, sulmazole and UD-CG 212.Cl. Similarly the negative inotropic effect of carbachol was antagonized concentration-dependently by saterinone. It is concluded that the new inotropic agents bind to A₁ adenosine receptors and that their interaction is of antagonist nature. This mechanism might contribute to their capacity to enhance force of contraction by stimulation of cAMP-formation in addition to phosphodiesterase inhibition. The effects of saterinone may be partially due to antagonism at m-cholinoceptors. This is presumably not the case with the other inotropic agents studied given their low affinity for this receptor.Send offprint requests to M. Böhm at the above addressSupported by the Deutsche Forschungsgemeinschaft     

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.     

Effects of trapidil-derivatives on calcium channel currents in isolated ventricular cells from mice

Summary A single glass micropipette voltage clamp technique with intracellular dialysis was used to study the effects of the trapidil derivatives AR 12–456 and AR 12–463 on Ca channel currents carried by Bat+ in isolated ventricular cells from mice hearts. Inspite of a more potent inhibition of the cAMP phosphodiesterase from heart (Bartel et al. 1985) a reversible Ca channel blocking action of both compounds could be observed. The concentration of half maximal block was calculated to about 50 mol/l for both derivatives tested. Neither a shift in the current-voltage relationships nor a significant change in the potential for half maximal activation was found. The maximal Ba²⁺ -conductance was reduced. The steady state inactivation was shifted towards more negative potentials by application of 100 mol/l AR 12–463. The decay of the Ba currents was accelerated in the range of the applied test potentials between –20 and +20 mV. It is concluded that the new trapidil derivatives with more potent inhibitory action on cardiac phosphodiesterase than trapidil can block myocardial Ca channels. Send offprint requests to B. Nilius     

Multiple effects of caffeine on calcium current in rat ventricular myocytes

Caffeine exerts a number of different effects on L-type calcium current in rat ventricular myocytes. These include: (1) a slowing of inactivation that is comparable to, but not additive to, that produced by prior treatment of the cells with ryanodine (a selective sarcoplasmic reticulum Ca²⁺ releaser) or high concentrations of intracellular 1,2-bis[2-aminophenoxy]ethane-N,N,N,N-tetraacetic acid (BAPTA) (a fast Ca²⁺ chelator), (2) a stimulation of peak I _Ca that is comparable to, but not additive to that produced by prior treatment with isobutylmethylxanthine (a selective phosphodiesterase inhibitor), and (3) a dose-dependent decrease of peak I _Ca that is not prevented by pretreatment with any of these agents. None of the caffeine actions could be mimicked or prevented by administration of 8-phenyltheophylline, a specific adenosine receptor antagonist. We conclude that only the slowing of I _Ca inactivation is due to caffeine's ability to deplete the sarcoplasmic reticulum of calcium. The stimulatory effect of caffeine on peak I _Ca is probably due to phosphodiesterase inhibition, while caffeine's inhibitory effect on I _Ca is independent of these processes and could be a direct effect on the channel. The multiplicity of caffeine actions independent of its effects on the sarcoplasmic reticulum lead to the conclusion that ryanodine, though slower acting and essentially irreversible, is a more selective agent than caffeine for probing sarcoplasmic reticulum function and its effects on other processes.The experimental part of this work was published during the postdoctoral stay of I. Zahradník in the Department of Physiology and Biophysics, The University of Texas Medical Branch, Galveston, TX 77555, USA     

Additive Antinociception between Intrathecal Sildenafil and Morphine in the Rat Formalin Test

The possible characteristics of spinal interaction between sildenafil (phosphodiesterase 5 inhibitor) and morphine on formalin-induced nociception in rats was examined. Then the role of the opioid receptor in the effect of sildenafil was further investigated. Catheters were inserted into the intrathecal space of male Sprague-Dawley rats. For induction of pain, 50 µL of 5% formalin solution was applied to the hind-paw. Isobolographic analysis was used for the evaluation of drug interaction between sildenafil and morphine. Furthermore, naloxone was intrathecally given to verify the involvement of the opioid receptor in the antinociception of sildenafil. Both sildenafil and morphine produced an antinociceptive effect during phase 1 and phase 2 in the formalin test. The isobolographic analysis revealed an additive interaction after intrathecal delivery of the sildenafil-morphine mixture in both phases. Intrathecal naloxone reversed the antinociception of sildenafil in both phases. These results suggest that sildenafil, morphine, and the mixture of the two drugs are effective against acute pain and facilitated pain state at the spinal level. Thus, the spinal combination of sildenafil with morphine may be useful in the management of the same state. Furthermore, the opioid receptor is contributable to the antinocieptive mechanism of sildenafil at the spinal level.     

Modulation of guanylate cyclase and phosphodiesterase by monovalent cations and nucleoside triphosphates in light-sensitive excised patches of rod outer segments

Excised inside-out patches of vertebrate rod outer segment can support phototransduction. I have examined how ionic and metabolic conditions influence the functional properties of light-sensitive patches fromGekko gekko. I find that such patches retain a variable level of basal phosphodiesterase activity, which lowers the cyclic guanosine monophosphate (cGMP) concentration reaching the channels and reduces the dark current. The dose/response relationship for channel opening by cGMP varies among patches and this variability is only reduced by working in darkness with the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX), suggesting that it is only partially due to phosphodiesterase activity. MgATP or MgGTP, but not Mg or ATP separately, increase this activity but a kinase does not appear to be involved. Intracellular monovalent cations also influence dark current intensity and light response kinetics. With 5 mM MgGTP, 1 mM IBMX, and 144 mM Li⁺, Na⁺, K⁺, or Rb⁺, dark current intensity and recovery time follow the respective sequences K⁺>Rb⁺>Na⁺>Li⁺ and K⁺<Rb⁺<Li⁺<Na⁺. Without IBMX, a dark current develops with K⁺ but not with Na⁺. These effects are not due to altered channel permeability (P) = 0.841.00 1.011.090.42], or differential Mg²⁺ block, but to modulation of guanylate cyclase, which overcomes phosphodiesterase when the major cation is K⁺ but not when it is Na⁺.     

Mechanisms of antagonistic action of internal Ca2+ on serotonin-induced potentiation of Ca2+ currents in Helix neurones

The influence of internal Ca²⁺ ions has been investigated during intracellular perfusion of isolated neurones from pedal ganglia of Helix pomatia in which serotonin (5-HT) induces a cyclic-adenosine-monophosphate-(cAMP)-dependent enhancement of high-threshold Ca²⁺ current (I _Ca). Internal free Ca²⁺ ([Ca²⁺]_i) was varied between 0.01 and 10 M by addition of Ca²⁺-EGTA [ethylenebis(oxonitrilo)tetraacetate] buffer. Elevation of [Ca²⁺]_i depressed the 5-HT effect. The dose/ effect curve for the Ca²⁺ blockade had a biphasic character and could be described by the sum of two Langmuir's isotherms for tetramolecular binding with dissociation constants K _d1=0.063 M and K _d2=1 M. Addition of calmodulin (CM) antagonists (50 M trifluoperazine or 50 M chlorpromazine), phosphodiesterase (PDE) antagonists [100 M isobutylmethylxanthine (IBMX) or 5 mM theophylline] and protein phosphatase antagonists [2 M okadaic acid (OA)] in the perfusion solution caused anticalcium action and modified the Ca²⁺ binding isotherm. Using the effect of OA and IBMX, two components of the total Ca²⁺ inhibition were separated and evaluated. In the presence of one of these blockers tetramolecular curves with K _d1=0.04 M and K _d2=0.69 M were obtained describing the activation of the retained unblocked enzyme — PDE or calcineurin (CN) correspondingly. The sum of these isotherms gave a biphasic curve similar to that in control. Leupeptin (100 M), a blocker of Ca²⁺-dependent proteases did not influence the amplitude of 5-HT effect, indicating that channel proteolysis is not involved in the depression. Our findings show that the molecular mechanism of Ca²⁺-induced suppression of the cAMP-dependent upregulation of Ca²⁺ channels is due to involvement of two Ca²⁺-CM-dependent enzymes: PDE reducing the cAMP level, and CN causing channel dephosphorylation. No other processes are involved in the investigated phenomenon at a Ca²⁺ concentration of less than or equal to 10 M.     

Rolipram forms a potent discriminative stimulus in drug discrimination experiments in rats

Long Evans rats were trained to discriminate 0.2 mg/kg IP (±)-rolipram from vehicle injection in a food-motivated two-lever operant task. Eight out of nine rats acquired the discrimination after an average of 91 sessions (min 65, max 137). The ED₅₀ of (±)-rolipram was 0.06 mg/kg IP. Generalization tests with (–)- and (+)-rolipram showed that the (–)-isomer was 8 times more active than (+)-rolipram with an ED₅₀ of 0.06 and 0.4 mg/kg IP respectively. The phosphodiesterase inhibitor RO 20-1724 partially (83%) generalized to (±)-rolipram in doses of 0.6 and 1.0 mg/kg IP. IBMX 5 mg/kg IP showed 63% generalization. Tests with imipramine and the (+)- and (–)-isomer of the noradrenaline uptake inhibitor oxaprotiline suggest that NA-uptake inhibiting drugs do not form an interoceptive cue which is (±)-rolipram-like. dbcAMP 12.5 mg/kg SC and 100 mg/kg SC dbcGMP did not generalize to the training drug. The nature of the discriminative stimulus produced by this dose of (±)-rolipram in rats remains to be elucidated. Offprint requests to: R. Ortman     

Contribution of phosphodiesterase isozymes to the regulation of the L-type calcium current in human cardiac myocytes

1. To determine the contribution of the various phosphodiesterase (PDE) isozymes to the regulation of the L-type calcium current (I_Ca(L)) in the human myocardium, we investigated the effect of selective and non-selective PDE inhibitors on I_Ca(L) in single human atrial cells by use of the whole-cell patch-clamp method. We repeated some experiments in rabbit atrial myocytes, to make a species comparison.
2. In human atrial cells, 100 μM pimobendan increased I_Ca(L) (evoked by depolarization to +10 mV from a holding potential of −40 mV) by 250.4±45.0% (n=15), with the concentration for half-maximal stimulation (EC₅₀) being 1.13 μM. I_Ca(L) was increased by 100 μM UD-CG 212 by 174.5±30.2% (n=10) with an EC₅₀ value of 1.78 μM in human atrial cells. These two agents inhibit PDE III selectively.
3. A selective PDE IV inhibitor, rolipram (1–100 μM), did not itself affect I_Ca(L) in human atrial cells. However, 100 μM rolipram significantly enhanced the effect of 100 μM UD-CG 212 on I_Ca(L) (increase with UD-CG 212 alone, 167.9±33.9, n=5; increase with the two agents together, 270.0±52.2%; n=5, P<0.05). Rolipram also enhanced isoprenaline (5 nM)-stimulated I_Ca(L) by 52.9±9.3% (n=5) in human atrial cells.
4. In rabbit atrial cells, I_Ca(L) at +10 mV was increased by 22.1±9.0% by UD-CG 212 (n=10) and by 67.4±12.0% (n=10) by pimobendan (each at 100 μM). These values were significantly lower than those obtained in human atrial cells (P<0.0001). Rolipram (1–100 μM) did not itself affect I_Ca(L) in rabbit atrial cells. However, I_Ca(L) was increased by 215.7±65.2% (n=10) by the combination of 100 μM UD-CG 212 and 100 μM rolipram. This value was almost 10 times larger than that obtained for the effect of 100 μM UD-CG 212 alone.
5. These results imply a species difference: in the human atrium, the PDE III isoform seems dominant, whereas PDE IV may be more important in the rabbit atrium for regulating I_Ca(L). However, PDE IV might contribute significantly to the regulation of intracellular cyclic AMP in human myocardium when PDE III is already inhibited or when the myocardium is under β-adrenoceptor-mediated stimulation.