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

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

The differential effect of calcium antagonists on the positive inotropic effects induced by calcium and monensin in cardiac preparations of rats and guinea-pigs

Summary It was the aim of the present study to gain more insight into the role of extracellular calcium and of calcium from intracellular sources in the development of contractile force in the mammalian heart. In rat Langendorff hearts the effect of nifedipine, verapamil, diltiazem, bepridil and lidoflazine as well as of the intracellularly acting calcium antagonists ryanodine and TMB-8 on the increase of the left ventricular pressure induced by calcium and the sodium ionophore monensin, respectively, was studied. In rat and guinea-pig papillary muscles the influence of nifedipine, ryanodine and lidoflazine on the effect of monensin on the force of contraction was evaluated.Calcium and monensin concentration-dependently increased the left ventricular pressure in rat Langendorff hearts. The calcium-induced effect was characterized by a sharp initial rise of the left ventricular pressure which stabilized at a lower level while monensin elicited a gradual rise of the left ventricular pressure. Nifedipine, verapamil and diltiazem, applied at the EC₅₀ and the EC₈₀ for the reduction of the left ventricular pressure under control conditions, shifted the concentration-response curves for calcium and monensin into the right. Ryanodine, TMB-8, lidoflazine and bepridil, applied at the EC₅₀, displaced the concentration-response curves for calcium and monensin to the right but reduced the maximal increase of the left ventricular pressure. At the EC₈₀, these drugs almost completely abolished the positive inotropic effects elicited by calcium and monensin, respectively. In rat papillary muscles monensin did not influence the basal force of contraction. A clear positive inotropic effect was only observed in the presence of nifedipine. In guinea-pig papillary muscles monensin increased the force of contraction. This effect was not influenced by nifedipine. Lidoflazine and ryanodine suppressed the monensin-induced force of contraction by 40 and 70%, respectively.In rat Langendorff hearts differential mechanisms underlie the calcium and monensin-induced increase of the left ventricular pressure. However, the inhibitory effect of ryanodine shows that the sarcoplasmic reticulum is instrumental for the development a positive inotropic effect by calcium and monensin. Influx of calcium through dihydropyridine-sensitive channels appears not to be directly involved.In addition to calcium entry blockade, bepridil and lidoflazine seem to act at an intracellular site. These drugs may influence the release of calcium from the sarcoplasmic reticulum or directly inhibit the contractile apparatus.The unmasking of the monensin-induced positive inotropic effect in rat but not in guinea-pig papillary muscles by nifedipine further illustrates the importance of dihydropyridine-sensitive calcium channels in the rat heart and their role to supply the sarcoplasmic reticulum with calcium.Preliminary results have been presented at the Spring Meeting of the German Pharmacological and Toxicological Society, 10–13 March, 1987, Mainz (Hugtenburg and Van Zwieten 1987)     

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 selective phosphodiesterase inhibitors on platelet-activating factor- and antigen-induced airway hyperreactivity, eosinophil accumulation, and microvascular leakage in guinea pigs

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 g 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 g 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.     

Potent inhibitory action of chlorethylclonidine on the positive inotropic effect and phosphoinositide hydrolysis mediated via myocardial alpha1-adrenoceptors in the rabbit ventricular myocardium

Summary The influence of the alpha_lb-adrenoceptor-selective antagonist chlorethylclonidine on the alpha₁-adrenergic positive inotropic effect and the phosphoinositide hydrolysis induced by phenylephrine was investigated in the rabbit ventricular myocardium. Pretreatment of membrane fractions derived from the rabbit ventricular muscle with 10^–5 mol/l chlorethylclonidine decreased the specific binding of [³H]prazosin (at a saturating concentration of 10^–9 mol/l) from the control value of 11.27±0.48 to 4.18±1.87 fmol/mg protein. The inhibition by adrenaline of the binding of [³H]prazosin (slope factor and affinity) was not affected by chlorethylclonidine. The positive inotropic effect of phenylephrine (in the presence of 3 × 10^–7 mol/l bupranolol) was inhibited by chlorethylclonidine in a concentration-dependent manner (10^–7–10^–5 mol/l) and abolished by 10^–5 mol/l chlorethylclonidine. The concentration of chlorethylclonidine to inhibit the phenylephrine-induced maximum response to 50% was 2.4 × 10^–6 mol/l. The accumulation of [³H]inositol monophosphate and [³H]inositol trisphosphate induced by 10^–5 mol/l phenylephrine was inhibited by chlorethylclonidine in the same concentration range. These findings indicate that the myocardial alpha₁-adrenoceptors mediating a positive inotropic effect in the rabbit ventricular myocardium may belong to the chlorethylclonidine-sensitive alpha_1b-subtype, and that the subcellular mechanism of action involve phosphoinositide hydrolysis. Send offprint requests to M. Endoh at the above address     

Effects of a cardiotonic quinolinone derivative Y-20487 on the isoproterenol-induced positive inotropic action and cyclic AMP accumulation in rat ventricular myocardium: comparison with rolipram, Ro 20-1724, milrinone, and isobutylmethylxanthine.

The effect of a new cardiotonic agent Y-20487 [6-(3,6-dihydro-2-oxo-2H-1,3,4-thiadiazin-5-yl)-3,4-dihydro-2(1H)- quinolinone] on cyclic AMP levels of rat ventricular cardiomyocytes and the contractile force of papillary muscles was investigated in comparison with selective cyclic AMP phosphodiesterase (PDE) inhibitors, milrinone (PDE-III selective), rolipram and Ro 20-1724 (PDE-IV selective), and a nonselective inhibitor 3-isobutyl-1-methylxanthine (IBMX). Rolipram and Ro 20-1724 did not elicit cyclic AMP accumulation and positive inotropy, but they potentiated the isoproterenol (ISO)-induced cyclic AMP accumulation more effectively than IBMX. Rolipram was more effective than Ro 20-1724 in enhancing ISO-induced cyclic AMP accumulation but was less effective in enhancing the ISO-induced positive inotropic effect, indicating that these agents produce a differential action on cyclic AMP metabolism and inotropy. Milrinone and Y-20487 elicited cyclic AMP accumulation and positive inotropy by themselves. Whereas milrinone scarcely affected the ISO-induced effects, Y-20487 shifted the concentration-response curve for the positive inotropic effect of ISO to the left to the same extent that IBMX did. Y-20487, however, was much less effective than IBMX in enhancing the ISO-induced cyclic AMP accumulation. The present results indicate that in rat ventricular myocardium, PDE-IV may play a crucial role in breakdown of cyclic AMP generated by beta-adrenoceptor stimulation, whereas other types of PDE isoenzymes, including PDE-III, may be responsible for the cyclic AMP accumulation and direct positive inotropic effect induced by PDE inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhancement of amrinone-induced positive inotropy in rabbit papillary muscles with depressed contractile function: effects on cyclic nucleotide levels and phosphodiesterase isoenzymes.

The inotropic activity of amrinone and its effects on cyclic nucleotide levels in rabbit papillary muscles with normal and depressed contractile function have been compared. The effects of amrinone on the cyclic (c) AMP hydrolytic activity of cyclic nucleotide phosphodiesterase (PDE) isoenzymes were also examined. Amrinone (2.4 x 10(-4) - 1.2 x 10(-3) M) produced a relatively weak (maximal increase 11%) positive inotropic effect in papillary muscles stimulated at the near optimal stimulation frequency of 1 Hz. In contrast, large positive inotropic responses (maximal 138-200%) were obtained with amrinone in papillary muscles in which contractile force had been depressed by: (a) lowering stimulation frequency to 0.4 Hz, (b) reducing extracellular Ca2+ concentration from 2.5 x 10(-3) M to 6.3 x 10(-4) M, (c) prior addition of sodium pentobarbitone (6.5 x 10(-4) M). The EC50 values for amrinone under conditions (a), (b), and (c) were 3.0 x 10(-3), 2.6 x 10(-3), and 2.8 x 10(-3) M, respectively. Force-frequency curves in rabbit papillary muscles were compared at normal (2.5 x 10(-3) M) and low (6.3 x 10(-4) M) extracellular Ca2+ concentration. Contractions at low frequencies of stimulation (less than 0.4 Hz) were less sensitive to removal of extracellular Ca2+ than higher stimulation rates indicating that in the former situation, recycling of intracellular Ca2+ is more important for maintaining contractile force.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Pharmacological differentiation of synergistic contribution of L-type Ca2+ channels and Na+/H+ exchange to the positive inotropic effect of phenylephrine,endothelin-3 and angiotensin II in rabbit ventricular myocardium

The present study was designed to delineate pharmacologically the role of sarcolemmal L-type Ca²⁺ channels and Na⁺/H⁺ exchange in the positive inotropic effect (PIE) of phenylephrine mediated by alpha-1 adrenoceptors, endothelin (ET) and angiotensin II (Ang II) that stimulate phosphoinositide (PI) hydrolysis in the rabbit ventricular muscle. The PIE of these receptor agonists was compared with the PIE of isoprenaline that accumulates cyclic AMP. For this purpose, we investigated the influence of a Ca²⁺ antagonist, verapamil, and of an inhibitor of Na⁺/H⁺ exchange, 5-(N-ethyl-N-isopropyl) amiloride (EIPA), alone or in combination, on the cumulative concentration-response curve (CRC) for phenylephrine (with 0.3 μM bupranolol), ET-3 and Ang II in isolated right ventricular papillary muscles of the rabbit, which were electrically stimulated at 1 Hz in Krebs-Henseleit solution at 37°C. Verapamil at 0.3 and 1 μM decreased the basal force of contraction to 37.0 ± 4.0% and 13.2 ± 1.1% of the control, respectively, while EIPA even at 10 μM affected the basal force to much less extent and decreased it to 87.0 ± 1.4%. Verapamil (0.3 and 1 μM) and EIPA (1 and 10 μM), when used alone, each significantly attenuated but did not abolish the PIEs induced by phenylephrine, ET-3 and Ang II, while the simultaneous administration of verapamil (1 μM) and EIPA (10 μM) consistently and almost completely inhibited the PIE induced by these receptor agonists. By contrast, the PIE of isoprenaline was retained even in the presence of verapamil and EIPA. These results indicate that both the influx of Ca²⁺ ions through L-type Ca²⁺ channels and activation of Na⁺/H⁺ exchange contribute synergistically to the PIE that is mediated by alpha-1 adrenergic, ET and Ang II receptor agonists, while these mechanisms are not essential for the beta-adrenoceptor-mediated PIE. Received: 20 February 1996 / Accepted: 20 August 1996     

Role of alpha1A adrenoceptor subtype in production of the positive inotropic effect mediated via myocardial alpha,adrenoceptors in the rabbit papillary muscle: influence of selective alpha1A subtype antagonists WB 4101 and 5-methylurapidil

Summary In order to elucidate the contribution of alpha_1A subtype to the positive inotropic effect mediated by myocardial alpha, adrenoceptors, the influence of the alpha_1A selective antagonists WB 4101 and 5-methylurapidil on the alpha,-mediated positive inotropic effect (induced by phenylephrine in the presence of a beta adrenoceptor blocking agent bupranolol) was assessed in the isolated rabbit papillary muscle. WB 4101 (10^–9-10^–7mol/l) shifted the concentration-response curve of the alpha,-mediated positive inotropic effect to the right in parallel, but the slope of Schild plot did not meet the competitive antagonism: WB 4101 shifted the curve by log one unit at 10^–9 mol/1, whereas it did not cause further shift at higher concentrations of 10^–8 and 10^–7 mol/l. WB 4101 did not affect the beta adrenoceptor-mediated positive inotropic effect. 5-Methylurapidil (10^–9 to 10^–7 mol/l) shifted the curve of alpha₁-mediated positive inotropic effect to the right and downwards in a concentration-dependent manner; the slope of Schild plot calculated at the level of 20% of the maximum response to phenylephrine was close to unity. 5-Methylurapidil at 3 × 10^–7 mol/1 abolished the alpha₁-mediated positive inotropic effect. In addition, 5-methylurapidil inhibited the beta adrenoceptor-mediated positive inotropic effect in the same concentration range as it antagonized the alpha₁-mediated positive inotropic effect, indicating that 5-methylurapidil is not selective for myocardial alpha, adrenoceptors. In the membrane fraction derived from the rabbit ventricular muscle, 5-methylurapidil displaced the specific binding of [³H]CGP-12177 with high affinity, whereas WB 4101 did not affect the [³H]CGP-12177 binding in the concentration range that it antagonized the alpha,-mediated positive inotropic effect. The present results indicate that alpha_1A adrenoceptor subtype plays a role in production of the positive inotropic effect mediated by myocardial alpha, adrenoceptors, but the extent is less than that mediated by alpha_1B subtype in the rabbit ventricular myocardium. Send offprint requests to M. Endoh at the above address     

Labelling of I2B-imidazoline receptors by [3H]2-(2-benzofuranyl)-2-imidazoline (2-BFI) in rat brain and liver: characterization, regulation and relation to monoamine oxidase enzymes

The novel selective imidazoline radioligand [3H]2-(2-benzofuranyl)-2-imidazoline (2-BFI) was used to characterize and assess further the nature of I₂-imidazoline receptors in rat brain and liver. In the cerebral cortex, 2-BFI displayed high affinity (K _i= 9.8 nM) for a single class of [³H]2-BFI binding sites. Other imidazoline/guanidine compounds (e.g. aganodine, cirazoline and idazoxan) displayed biphasic competition curves, indicating the existence of high (K _iH= 2.9-78 nM; R _H= 61-83%) and low (K _iL= 4.7-158 μM) affinity sites. The pharmacological profile for [³H]2-BFI binding (aganodine > cirazoline > 2-BFI >> clonidine > amiloride >> efaroxan) was typical of that for I₂-sites. This profile was almost identical to that obtained against [³H]idazoxan (correlation between pK _i values, r = 0.97) which indicated that the sites characterized with [³H]2-BFI in brain corresponded to I₂-imidazoline receptors. The low affinity of amiloride against [³H]2-BFI (K _i= 900 nM) further indicated that these brain I₂-sites belong to the I_2B-subtype. [³H]2-BFI binding sites (B _max= 72 fmol/mg protein) in brain were differentially modulated by treatment (7 days) with cirazoline (up-regulation: 25%) and the MAO inhibitor phenelzine (down-regulation: 31%), indicating that these I₂-sites are regulated in vivo, as is the case for those labelled by [³H]idazoxan. Chronic treatment with 2-phenylethylamine, a phenelzine metabolite and endogenous amine, did not alter the density of brain of I₂-imidazoline receptors labelled by [³H]idazoxan. Preincubation of liver membranes with the MAO inhibitor clorgyline (10^-7 M) abolished the binding of [³H]Ro 41-1049 (N-(2-aminoethyl)-5-(m-fluorophenyl)-4-thiazole carboxamide) to MAO-A, but it did not alter the binding of [³H]Ro 19-6327 (N-(2-aminoethyl)-5-chloro-2-pyridine carboxamide) to MAO-B or that of [³H]2-BFI to I₂-sites. At 10^-4 M it also abolished MAO-B sites, but a substantial proportion of I₂-sites (40%) remained intact. Preincubation of liver membranes at 60 °C also abolished MAO-A/B sites, whereas still 22% of I₂-sites remained. The results indicate that [³H]2-BFI is a good tool for the identification of I₂-imidazoline receptors and suggest further that certain I₂-sites and MAO are different proteins. Received: 10 January 1997 / Accepted: 6 March 1997