High selectivity for inhibition of phosphodiesterase III and positive inotropic effects of MCI-154 in guinea pig myocardium.

MCI-154 (0.3-100 microM) exerted a concentration-dependent positive inotropic effect in isolated guinea pig papillary muscles (EC50 0.8 microM). The efficacy of MCI-154 (253% of predrug value) was 1.7-fold higher than that of saterinone but comparable to that of milrinone. Carbachol markedly reduced the increase in force of contraction (FOC) of MCI-154. In intact contracting papillary muscles, the positive inotropic effect was accompanied by an increase in cyclic AMP content to 0.78 +/- 0.09 pmol/mg wet weight (n = 10), corresponding to 150% of the basal value (0.51 +/- 0.05 pmol/mg wet weight, n = 21) in the presence of submaximal cyclic AMP phosphodiesterase (PDE) isoenzyme III inhibiting concentrations of MCI-154 (30 microM). MCI-154 (1-1,000 microM) concentration-dependently inhibited the activity of PDE III from homogenates of guinea pig myocardium. The IC50 was 3.8 microM. PDE I, II, and IV were not significantly affected up to 100 microM (PDE I and IV) and up to 1,000 microM (PDE II). In comparison, milrinone and saterinone were PDE III/IV-selective PDE inhibitors. Rolipram inhibited PDE IV only. IBMX and theophylline were nonselective PDE inhibitors. MCI-154 had only a marginal positive chronotropic effect. The frequency of spontaneously beating right auricles from guinea pig heart was increased by 8.7% at most (n = 5). MCI-154 increased Ca2+ sensitivity in chemically skinned porcine ventricular muscle fibers.(ABSTRACT TRUNCATED AT 250 WORDS)

Very high affinity interaction of DPI 201-106 and BDF 8784 enantiomers with the phenylalkylamine-sensitive Ca2(+)-channel in Drosophila head membranes.

1. Piperazinylindoles (DPI 201-106, BDF 8784), drugs known to act on voltage-dependent Na(+)-channels, bind with very high affinity to a Ca2(+)-channel-associated phenylalkylamine receptor in Drosophila melanogaster head membranes. These compounds and (+)-tetrandrine, a naturally occurring Ca2(+)-antagonist, were the most selective inhibitors for phenylalkylamine-labelled Drosophila Ca2(+)-channels compared to mammalian L-type Ca2(+)-channels. 2. Replacement of the cyano group by a methyl group in (+)-DPI 201-106 ((+)-BDF 8784) increases the IC50 value for inhibition of phenylalkylamine labelling of Drosophila Ca2(+)-channels from 0.29 to 2.1 nM but decreases the IC50 value for inhibition of phenylalkylamine labelling of mammalian skeletal muscle Ca2(+)-channels from 3480 to 49 nM. 3. DPI 201-106 enantiomers completely block (at 0.1 microM) phenylalkylamine photolabelling of a 136 K polypeptide in Drosophila head membranes whereas 10 microM aconitine or lidocaine are without effect. 4. Assessment of the Ca2(+)-antagonist effects of the substituted DPI 201-106 enantiomers in K(+)-depolarized taenia strips from guinea-pig caecum yielded pA2 values of 6.33 +/- 0.07 for (-)-BDF 8784 and 6.99 +/- 0.17 for (+)-BDF 8784, respectively. 5. Piperazinylindoles, previously believed to act nonspecifically on voltage-dependent mammalian L-type Ca2(+)-channels, therefore have stereoselectivity for a novel binding site and chemical selectivity unrelated to local anaesthetic activity. 6. It is proposed that a very high affinity piperazinylindole-selective site is coupled to the phenylalkylamine receptor of Drosophila Ca2(+)-channels. These sites are still present on mammalian L-type Ca2(+)-channels but have lower affinity and/or are less tightly coupled to phenylalkylamine receptors on the alpha 1-subunit.

Effect of temperature on alpha 2- and alpha 1-adrenoceptor-mediated responses in the pithed rat and in the rat vena cava.

1. The influence of body temperature on the alpha 1- and alpha 2-adrenoceptor-mediated pressor responses has been investigated in the pithed rat. 2. The pressor responses to noradrenaline, to the full alpha 1-adrenoceptor agonists phenylephrine and cirazoline and to tyramine were not influenced by lowering the temperature from 36-37 degrees C to 27-29 degrees C. In contrast, the dose-response curves for the pressor effects of the partial alpha 1-adrenoceptor agonist ST 587 and of the alpha 2-adrenoceptor agonist B-HT 920 (2-amino-6-allyl-5,6,7,8-tetrahydro-4H-thiazolo-[4,5-d]-azepine), B-HT 933 (2-amino-6-ethyl-5,6,7,8-tetrahydro-4H-[4,5-d]azepine), clonidine, moxonidine and M-7 (2-dimethylamino-5,6-dihydroxy-1,2,3,4-tetrahydronaphthalene hydrobromide) were markedly depressed (without change in the ED50 values), when the body temperature was lowered from 36-37 degrees C to 27-29 degrees C. 3. After i.v. administration of yohimbine, there was a rightward shift of the dose-response curve for B-HT 920, and the degree of this shift was the same at all temperatures investigated. 4. In the rat vena cava preincubated with [3H]-noradrenaline, the B-HT 920-induced alpha 2-adrenoceptor-mediated inhibition of electrically evoked tritium overflow was also reduced at lower temperature. 5. These results are compatible with the suggestion that cooling decreases the alpha 2-adrenoceptor-mediated pre- and postsynaptic responses in the rat vena cava and pithed rat respectively, leaving the pressor effect induced by full, but not partial alpha 1-adrenoceptor agonists in the pithed rat unaffected. 6. These differences may partly be related to differences in receptor reserve for alpha 1- and alpha 2-adrenoceptors in the pithed rat preparation.

Central presynaptic alpha 2-autoreceptors are involved in the blood pressure-lowering effect of moxonidine.

The alpha-adrenoceptor-mediated effects of the newly developed antihypertensive drug moxonidine and of clonidine were compared in four in vitro models and in anesthetized rabbits. Inhibition of the electrically induced twitch response in the isolated rat vas deferens by moxonidine and clonidine (pEC50 values 7.8 and 8.5) was counteracted by yohimbine. The maximum degree of inhibition obtained with both drugs was identical. The contractile response of the rat anococcygeal muscle to moxonidine and clonidine (pEC50 6.2 and 6.9) was antagonized by prazosin. The maximum degree of contraction obtained with moxonidine was similar in degree to that of phenylephrine, whereas that of clonidine was lower. Moxonidine and clonidine inhibited the electrically evoked [3H]norepinephrine (NE) overflow in strips of rabbit pulmonary artery (pEC50 7.1 and 6.6) and in rat brain cortex slices (pEC50 6.9 and 7.7); the effect of moxonidine was counteracted by rauwolscine. After intracisternal injection in anesthetized rabbits, moxonidine lowered blood pressure (BP) more potently than did clonidine. Intracisternal pretreatment of rabbits with 6-hydroxydopamine strongly attenuated the hypotensive effect of moxonidine without affecting that of clonidine. The hypotensive effect of moxonidine was abolished by pretreatment with reserpine and alpha-methyl-p-tyrosine (alpha-MPT). In conclusion, in isolated tissues of the rat, moxonidine is as selective as clonidine as an alpha-adrenoceptor agonist. The hypotensive effect of moxonidine, unlike that of clonidine, appears to involve central presynaptic alpha 2-autoreceptors in large part.

Sodium channel comodification with full activator reveals veratridine reaction dynamics.

Veratridine association and dissociation rates were determined at single sodium channels in outside-out patches of cultured ventricular myocytes obtained from late-fetal rat hearts. In single cardiac sodium channels depolarized from -110 to -30 mV, intracellular veratridine induced a long lasting (tau = 0.48 sec) open state with small current amplitude (-0.3 pA, i.e., 1/4 of normal) and frequent closing transitions, giving it a burstlike appearance, in agreement with reports on other types of sodium channel. Veratridine-associated and veratridine-free states of a single sodium channel were monitored by comodifying it with an allosteric activator, BDF 9145 (1 microM), that induced a burst with normal open channel current amplitude (-1.2 pA at -30 mV) upon veratridine dissociation. Veratridine and BDF 9145 interacted with reciprocal synergism at the single sodium channel such that veratridine-induced bursts (called P-bursts for partially activated) alternated with BDF 9145-induced bursts (called F-bursts for fully activated) many times following a single depolarization to -30 mV. P-bursts and F-bursts within such trains of bursts had exponentially distributed durations. The reciprocal time constant for F-bursts, tau F-1, increased linearly with veratridine concentration (0.3-30 microM), whereas tau P was insensitive. We conclude, therefore, that P-bursts reflect veratridine occupancy and F-bursts reflect the veratridine-free state; if veratridine and BDF 9145 bind to a sodium channel simultaneously, veratridine exerts conformational dominance, i.e., retains its property to reduce channel conductance. For the single cardiac sodium channel activated (i.e., deprived of inactivation) by BDF 9145, we have determined a veratridine association rate constant k1 = 4.3 x 10(6) M0-1 sec-1, dissociation rate constant K-1 = 2.2 sec-1 and equilibrium dissociation constant KD = 5.1 x 10(-7) M (20 degrees, -30 mV membrane potential).

Lack of stereoselectivity in the inotropic and phosphodiesterase inhibitory effects of saterinone enantiomers.

The enantiomers of the positive inotropic and a1-adrenoceptor blocking agent saterinone (+/-)-1,2-dihydro-5-[4-[2-hydroxy-3- [4-(2-methoxyphenyl)-1-piperazinyl]propoxy] phenyl]-6-methyl-2-oxo-3-pyridine-carbonitrile, BDF 8634) have been investigated with in vitro and in vivo models in laboratory animals. In the guinea pig papillary muscle, saterinone enantiomers had equipotent inotropic activity and were also as potent as racemic saterinone; the (R)-enantiomer, however, exhibited a greater efficacy than the related compounds. Saterinone and its enantiomers were equipotent in the inhibition of phosphodiesterase PDE III activity in the guinea pig myocardium. The equipotent inotropic effects were also observed after parenteral and enteral administration in cats. In receptor binding studies, (S)-saterinone was 10-fold more potent than (R)-saterinone by inhibiting [3H]-prazosin binding to specific alpha 1-adrenoceptor sites in rat brain cortex membranes. However, in the isolated thoracic aorta of the rabbit, (S)-saterinone was only 3-fold more potent than (R)-saterinone at preventing the pressor effects of phenylephrine. When the enantiomers were tested in vivo against the pressor effects of phenylephrine in the pithed rat, (S)-saterinone was only 2-fold more potent than (R)-saterinone in its alpha 1-adrenoceptor blocking potency. Thus the enantiomers of saterinone do not display enantio-selectivity in their inotropic and PDE III inhibitory effects in vitro, nor in their cardiotonic effects in vivo. There is a slight enantio-selectivity at alpha 1-adrenoceptors in receptor binding studies, but this is reduced to biologically irrelevant magnitude in functional studies in vitro and in vivo.

Reversal of the cardiotonic and action-potential prolonging effects of DPI 201-106 by BDF 8784, a methyl-indol derivative.

1. We studied the interaction of the cardiotonic compound DPI 201-106 (4-[3'-(4''-benzhydryl-1''-piperazinyl)-2'-hydroxypropoxy]-1H-indole-2- carbonitrile; DPI) and its derivative BDF 8784 (2-methyl-4-[3'-(4''-benzhydryl- 1''-piperazinyl)-2'-hydroxypropoxy]-1H-indole; BDF) in isolated right ventricular papillary muscles of guinea-pig heart. 2. In contrast to the cardiotonic DPI, the methyl-indole derivative lacked a positive inotropic effect and even caused negative inotropic effects in concentrations above 1 microM. At 10 microM BDF significantly reduced the force of contraction and dV/dtmax, but did not affect action potential duration (APD). 3. Pretreatment of papillary muscles with BDF prevented the positive inotropic action of DPI in a concentration-dependent, but non-competitive fashion. At 10 microM, BDF prevented the inotropic effect of racemic DPI and shortened the DPI-induced prolongation of action potential duration. BDF similarly affected the inotropic and APD-prolonging effects of the sea anemone polypeptide ATX II. 4. In cardiac myocytes, DPI induced a tetrodotoxin (TTX)-sensitive, slowly inactivating inward current. The slow decay of this current was enhanced by BDF. In cells pretreated with BDF, DPI was not effective. BDF alone depressed the sodium and the calcium current. 5. In conclusion, the non-inotropic methyl-indole derivative BDF interacts with DPI noncompetitively at the sodium channels to abolish the inotropic and APD-prolonging effects of DPI, emphasizing the importance of the substituent in position 2 of the indole moiety for this effect.

General pharmacology of the novel centrally acting antihypertensive agent moxonidine.

Moxonidine (4-chloro-N-(4, 5-dihydro-1H-imidazol-2-yl)-6-methoxy-2-methyl-5-pyrimidinamine, BDF 5895) reduces blood pressure and heart rate in rats with genetic hypertension (SHR/Okamoto) and in rats with renovascular hypertension (Goldblatt 1 k/1 c). The hypotensive action was also confirmed in renal-hypertensive dogs. The hypotensive action is preceded by a reduction in plasma noradrenaline concentration, thus reflecting a reduction in sympathetic activity. In anesthetized cats, administration of moxonidine into the vertebral artery induces a greater hypotensive effect than i.v. injection of same doses, indicating the central nervous system as the site of hypotensive action. Similar to clonidine, the hypotensive action of moxonidine is abolished by pretreatment of the animals with a selective alpha 2-antagonist. Direct application of moxonidine into the cisterna magna of anesthetized rabbits revealed a 10-fold greater hypotensive potency than clonidine, in contrast to i.v. application where moxonidine was 10-fold less potent than clonidine. At least 10-fold higher doses of moxonidine were needed to cause side effects (sedation, inhibition of gastric secretion), when compared with clonidine. Interruption of presynaptic noradrenergic pathways completely abolished the hypotensive action of moxonidine. Thus moxonidine is endowed with a specific central site of action, presumably by stimulating central presynaptic alpha 2-adrenoceptors. This specific central hypotensive action enables a greater dissociation between the antihypertensive effect on the one hand, and the side effects on the other.

Unique presynaptic alpha 2-receptor selectivity and specificity of the antihypertensive agent moxonidine.

The characteristics of the alpha-receptor activating property of the new antihypertensive agent moxonidine (4-chloro-N-(4, 5-dihydro-1H-imidazol-2-yl)-6-methyl-2-methyl-5-pyrimidinamine, BDF 5895) was studied using peripheral vasculature and brain membranes of various animals. Moxonidine exerted a full agonist effect in elevating diastolic blood pressure in the pithed rat. Activation of postsynaptic alpha 1- and alpha 2-receptors contribute to the vasoconstrictory effect in rats. In the vasculature of the rabbit, moxonidine was a full agonist at presynaptic alpha 2-receptors in inhibiting transmitter release induced by electrical stimulation of pulmonary artery strips. At postsynaptic sites, exogenously applied moxonidine was a full agonist at alpha 1-receptors in the isolated aorta, pulmonary artery and vena cava of the rabbit. Selectivity for alpha 2-receptors in the pulmonary artery was 106-fold. In rat brain membranes, moxonidine showed 288-fold greater selectivity for alpha 2-receptors, when the displacement of [3H]-rauwolscine was compared with the displacement of [3H]-prazosin. On the whole, clonidine exhibited greater potency than moxonidine on both alpha-receptor subtypes, but moxonidine consistently showed greater alpha 2-receptor selectivity than clonidine. In the guinea pig myocardium, moxonidine caused neither bradycardia nor tachycardia in the isolated right atrium and produced a negligible positive inotropic effect at 100 mumol/l in the isolated papillary muscle.

Characteristics of the binding of the antihypertensive agent moxonidine to alpha 2-adrenoceptors in rat brain membranes.

The characteristics of the alpha 2-adrenoceptor binding property of moxonidine (4-chloro-N-(4,5-dihydro-1H-imidazol-2-yl)-6-methoxy-2-methyl-5-pyrim idinamine, BDF 5895), a newly developed centrally acting antihypertensive agent, was investigated in receptor binding assays using [3H]-rauwolscine bound to rat cortex membranes. The results are as follows: 1. [3H]-Rauwolscine binds in a saturable manner to rat cerebral cortex membranes with a Bmax of 320 +/- 50 fmol/mg of protein and KD of 1.7 +/- 0.5 nmol/l. 2. The alpha 2-adrenoceptor antagonist yohimbine competes with a Ki value of 8.0 nmol/l and slope factor close to unity. (--)-Norepinephrine competes with an overall Ki-value of 50 nmol/l and slope factor of 0.61. Mg2+ concentrations of 10 mmol/l in the presence of 0.1 mmol/l 3'-guanylimidodiphosphate (Gpp(NH)p) (Mg2+/Gpp(NH)p) do not affect the yohimbine inhibition curve, but increase the (--)-norepinephrine Ki value to 2.8 mumol and the slope factor to unity. 3. Moxonidine competes with [3H]-rauwolscine binding with an overall Ki value of 460 nmol/l and slope factor of 0.72. Moxonidine binding inhibition was potentiated by Mg2+, but shifted to lower potency by Mg2+/Gpp(NH)p and 150 mmol/l NaCl. 4. Binding inhibition curves for (--)-norepinephrine and moxonidine fitted better to a 2-site model and could be interpreted in terms of high affinity site (with Ki value KH) and low affinity site (with Ki value KL). The effect of Mg2+/Gpp(NH)p and NaCl was to convert the high affinity sites to low affinity sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacological properties of the positive inotropic and alpha 1-adrenoceptor blocking agent saterinone.

The pharmacological properties of saterinone [+/-)-1,2-dihydro-5-[4-[2-hydroxy-3-[4-(2-methoxyphenyl)-1-piperazinyl] propoxy]phenyl]-6-methyl-2-oxo-3-pyridine-carbonitrile, BDF 8634) were investigated in isolated organs of the guinea pig and in human platelets. Saterinone was found to be a potent antagonist at vascular alpha 1-adrenoceptors with a pA2-value of 8.46 +/- 0.12. Besides its affinity for alpha 1-adrenoceptors saterinone exerted a positive inotropic effect in the isolated papillary muscle at an EC50-value of 3.2 X 10(-6)mol/l indicating 10-fold greater potency than milrinone. Comparable EC50-values were also found for the inotropic, chronotropic and bronchodilatory actions of the drug, indicating a common mechanism for these effects. The inotropic effects were not mediated by beta-adrenergic or H2-histaminergic receptors, but were shown to involve an elevation of myocardial cyclic adenosine monophosphate (cAMP) content. Saterinone also inhibited crude cAMP phosphodiesterase (PDE) activity in homogenates obtained from guinea pig right ventricles. The IC50-value for PDE-inhibition was 2.3 X 10(-5) mol/l and thus at a higher concentration than the inotropic effect. Saterinone was a potent inhibitor of human platelet aggregation induced by adenosine diphosphate, collagen and arachidonate. Against the latter agonist, saterinone was about 40-fold more effective than acetylsalicylic acid. In conclusion, saterinone exhibited a dual mechanism of action--direct inotropic effects in the myocardium and alpha 1-receptor blockade in the guinea pig vasculature.(ABSTRACT TRUNCATED AT 250 WORDS)

Electrophysiologic effects of saterinone and milrinone in the isolated guinea pig myocardium.

The positive inotropic agent milrinone and the newly synthesized compound saterinone [+/-)-1,2-dihydro-5-[4-[2-hydroxy-3-[4-(2-methoxyphenyl)-1-piperazinyl] propoxy]phenyl]-6-methyl-2-oxo-3-pyridine-carbonitrile, BDF 8634) dose-dependently increased the contractile force of guinea pig left atria. During a 90-min exposure, 10(-4) mol/l saterinone caused a continuous increase of the functional refractory period (FRP), while the initial positive inotropic effect faded gradually. No change of the FRP was observed with milrinone. Saterinone (10(-4) mol/l) also increased the action potential duration and the FRP of guinea pig papillary muscles, while milrinone had no influence on either parameter. Both milrinone and saterinone increased the amplitude, depolarization velocity and duration of slow response action potentials in K+-depolarized muscles. These effects appeared in the presence of tetrodotoxin or propranolol and could be reversed by carbachol. It is concluded that saterinone increases the force of contraction and the slow inward current by inhibiting cardiac phosphodiesterase. The increase of the FRP may be attributed to a decrease of the membrane K+ conductance.

Positive inotropic and vasodilatory actions of saterinone in vivo.

The cardiovascular actions of the newly developed inotropic and alpha 1-receptor blocking agent saterinone [+/-)-1,2-dihydro-5-[4-[2-hydroxy-3-[4-(2-methoxyphenyl)-1-piperazinyl] propoxy]phenyl]-6-methyl-2-oxo-3-pyridine-carbonitrile, BDF 8634) was investigated in small laboratory animals in vivo. Saterinone caused a direct inotropic effect in pithed guinea pigs without affecting heart rate. In the same animal species saterinone competitively antagonized the pressor effects of phenylephrine at inotropic doses. In conscious rabbits saterinone exerted dose-dependent increase in left ventricular dP/dtmax and in heart rate, whilst reducing arterial blood pressure in the same dose range. The drug dose-dependently antagonized phenylephrine as evidence of its alpha 1-receptor blocking effects in the conscious rabbit. The duration of alpha 1-receptor blockade was longer than the duration of inotropic effects. The onset of inotropic and vascular effects of saterinone was found to be simultaneous, when the drug was slowly infused into the femoral vein of anesthetized cats. The saterinone dose which caused a significant inhibition of the pressor effects of phenylephrine (comparable to prazosin) still caused a reduction of femoral perfusion pressure and systemic blood pressure in anesthetized cats pretreated with phenoxybenzamine. Thus in contrast to prazosin, which was rendered ineffective after phenoxybenzamine, saterinone possesses an additional mechanism for vasodilation. Saterinone exhibited good oral efficacy in spontaneously hypertensive rats and in conscious cats, in which an oral dose of 10-30 mg/kg significantly reduced arterial blood pressure or increased left ventricular dP/dtmax, respectively. Thus saterinone exerts in vivo direct positive inotropic and vasodilating effects.(ABSTRACT TRUNCATED AT 250 WORDS)