Increased sensitivity to α-adrenoceptor stimulation but intact purinergic and muscarinergic effects in prehypertensive cardiac hypertrophy of spontaneously hypertensive rats

Summary The effects of phenylephrine, isoprenaline and adenosine, (–)-N⁶-phenylisopropyladenosine (PIA) or carbachol alone and in the presence of isoprenaline on force of contraction were studied in isolated electrically driven papillary muscles of spontaneously hypertensive rats (SHR) and age-matched Wistar control rats. In SHR an increased heart to body weight ratio was observed when blood pressure was not yet elevated. During this stage of the syndrome (i.e. between the 27th and 35th day of life) phenylephrine was about 3.4 times more potent to increase force of contraction in SHR (mean EC₅₀: 2.8 mol l^–1) than in control rats (mean EC₅₀: 9.4 mol l^–1). The positive inotropic effect of isoprenaline was similar in SHR and control rats. Also no difference could be detected in the isoprenaline-antagonistic effect of adenosine, the adenosine receptor agonist PIA or carbachol. We conclude that an increased sensitivity to cardiac -adrenoceptor stimulation might be related to prehypertensive cardiac hypertrophy in SHR.     

Pharmacological characteristics of adenosine-induced inhibition of dog ventricular contractility: dependence on the pre-existing level of β-adrenoceptor activation

Summary Experiments were carried out to characterize the adenosine-induced negative inotropic effect in relation to the extent of -adrenoceptor activation in the isolated dog left ventricular myocardium. Adenosine and R-N⁶-phenylisopropyladenosine inhibited the positive inotropic effect of isoprenaline (10^–7 mol/1 and lower) about 20% of its maximal response, which was antagonized by an A1 adenosine receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine in a concentration-dependent manner. The negative inotropic effect of adenosine disappeared and that of R-N⁶-phenylisopro-pyl-adenosine decreased when the isoprenaline concentration was elevated to the level higher than 10^–7 mol/1. Adenosine deaminase (1.5 U/ml) that abolished the negative inotropic effect of adenosine enhanced the effect of R-N⁶-phenylisopropyladenosine, indicating that endogenous adenosine released by high isoprenaline concentration (10^–6 mol/1) modulates the interaction. The maximal response to adenosine and R-N⁶-phenylisopro-pyladenosine determined in the presence of 10^–7 mol/1 isoprenaline was 50% of that of carbachol which elicited the maximal inhibition even in the presence of 10^–6 mol/1 isoprenaline. The negative inotropic effects of R-N⁶-phenylisopropyladenosine and carbachol were additive to the maximal response equivalent to that of carbachol. The difference in the efficiency between the adenosine and muscarinic receptor agonists may be partly ascribed to the difference in densities of the respective receptors in the dog ventricular myocardium. The negative inotropic effect of R-N⁶-phenylisopropyladenosine in the presence of isoprenaline was associated with decrease in cyclic AMP levels elevated previously by isoprenaline. The elevation of cyclic AMP levels caused by isoprenaline (3 × 10^–7 mol/1) was abolished by R-N⁶-phenylisopro-pyladenosine (10^–4 mol/1), while the contractile response was reduced only by 30% with R-N⁶-phenylisopro-pyladenosine. In the absence of -adrenoceptor stimulation R-N⁶-phenylisopropyladenosine elicited a negative inotropic effect without changes in cyclic AMP levels, but this effect was less than 10% of the basal force of contraction. It is concluded that in the dog ventricular myocardium adenosine receptors play a role for the inhibitory regulation of contractility, which is influenced markedly by the pre-existing level of -adrenoceptor activation. Send offprint requests to M. Endoh at the above address     

Effects of cantharidin on force of contraction and phosphatase activity in nonfailing and failing human hearts.

1. The effect of the phosphatase inhibitor, cantharidin (3-300 microM) on force of contraction was studied in isolated electrically driven right ventricular trabeculae carneae from human myocardium. 2. The positive inotropic effect of cantharidin started at a concentration of 100 microM with a positive inotropic effect to 199% and to 276% of the predrug value in nonfailing and failing human hearts, respectively. 3. Under basal conditions the contraction time parameters were prolonged in human heart failure vs. nonfailing preparations. However, the positive inotropic effect of cantharidin did not affect contraction time parameters. Thus, time to peak tension, time of relaxation and total contraction time were not shortened by cantharidin in nonfailing and failing preparations. 4. The phosphatase activity was unchanged in preparations from failing hearts compared to nonfailing hearts. 5. Cantharidin inhibited phosphatase activity in a concentration-dependent manner. The IC50 value of cantharidin was about 3 microM in both nonfailing and failing human myocardium. 6. The positive inotropic effect of cantharidin was similar in nonfailing and failing human hearts, accompanied by a similar inhibitory effect of cantharidin on the phosphatase activity. The positive inotropic effect of cantharidin in failing hearts was as strong as the effect of isoprenaline in nonfailing hearts. 7. It is concluded that the treatment with a phosphatase inhibitor may offer a new positive inotropic modality for the treatment of human heart failure.     

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     

Dopamine D-2 receptors inhibit D-1 stimulated cyclic AMP accumulation in striatum but not limbic forebrain

Summary The effect of dopamine D-2 receptor activtion on dopamine D-1 stimulated cyclic AMP accumulation was investigated in slices of rat striatum and limbic forebrain (nucleus accumbens and tuberculum olfactorium). In striatal slices the dose-dependent increase in cyclic AMP accumulation due to dopamine (3–100 mol/1) was enhanced by selective D-2 receptor blockade using (–)-sulpiride (30 mol/1). In limbic slices the increase in cyclic AMP due to dopamine (3–50 mol/l) was unaffected by selective D-2 receptor blockade. The enhancement of cyclic AMP accumulation due to the selective D-1 agonist SKF 38393 (2,3,4,5-tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine; 1 gmol/1) in striatal slices was attenuated in the presence of the selective D-2 receptor agonist LY 171555 (quinpirole hydrochloride; 10 mol/l). This attenuation was in turn blocked by (–)-sulpiride (10 mol/1). In limbic slices LY 171555 (10 mol/l) had no effect on SKF 38393 (1 mol/l) stimulated cyclic AMP accumulation. Conversely muscarine receptor activation, using carbachol (10 mol/l), attenuated D-1 stimulated cyclic AMP accumulation in both striatum and limbic forebrain. Dopamine D-2 or muscarine receptor stimulation in either striatal or limbic slices did not attenuate cyclic AMP accumulation due to VIP (vasoactive intestinal polypeptide; 0.5 mol/l), isoprenaline (10 mol/l) or 2-chloroadenosine (100 mol/l). This suggests that in striatal slices, D-2 receptors mediate a selective inhibition of D-1 stimulated cyclic AMP accumulation, but that in the limbic forebrain D-2 receptors are unlikely to be coupled to D-1 receptor-linked adenylate cyclase. These data indicate a fundamental difference in the properties of D-2 receptor-effector coupling in these brain regions. Send offprint requests to S. R. Nahorski at the above address     

The ryanodine binding sarcoplasmic reticulum calcium release channel in nonfailing and in failing human myocardium

The ryanodine-sensitive Ca²⁺ release channel (RyaCRC) of the sarcoplasmic reticulum plays a key role in the intracellular Ca²⁺ handling in cardiomyocytes. Altered expression of the RyaCRC has been supposed to contribute to abnormal cellular Ca²⁺ handling and to myocardial dysfunction in dilated and ischemic cardiomyopathy. In the present study the ³H-ryanodine binding site in human myocardial homogenates was characterized and the density of the RyaCRC (which corresponds to the cardiac ryanodine receptor) was determined in nonfailing and in failing human myocardium.Homogenates were prepared from nonfailing left ventricular myocardium from the hearts of 5 organ donors (NF) and from failing myocardium from 14 explanted hearts of transplant recipients with end-stage heart failure resulting from dilated (DCM, n = 5) or ischemic (ICM, n = 9) cardiomyopathy. Radioligand saturation binding experiments revealed a specific, high-affinity ³H-ryanodine binding site (K_d-values: NF: 0.65±0.11 nmol/l, DCM: 0.66±0.09 nmol/l, ICM: 0.88±0.18 nmol/l; n.s.) in all preparations. Specific ³H-ryanodine binding depended on the free Ca²⁺ concentration in the assay. It was maximal at 3–100 mol/l Ca²⁺. The binding was inhibited by the RyaCRC antagonists ruthenium red (K_i-value: 0.32 [0.18–0.56] mol/l, n = 5) and Mg²⁺ (K_i-value: 2.95 [1.23–7.11] mmol/l, n = 5). The RyaCRC density was 103.5±11.9 fmol/mg protein in nonfailing myocardium. There was no significant change in the RyaCRC density in dilated or ischemic cardiomyopathy (112.4±17.1 and 122.7±13.9 fmol/mg protein) compared to nonfailing control myocardium.In summary, ³H-ryanodine binds specifically and with high-affinity to the RyaCRC in human myocardium. There is no change in the RyaCRC density in failing myocardium of patients with DCM or ICM in comparison to nonfailing controls.     

Electrophysiologic and inotropic effects of α-adrenoceptor stimulation in human isolated atrial heart muscle

Summary The effects of -adrenoceptor stimulation on force of contraction were investigated in human atrial heart muscle and compared with those of -adrenoceptor stimulation. The maximal positive inotropic effect produced by stimulation of -adrenoceptors with phenylephrine (in the presence of atenolol 10 mol/l) was significantly smaller than that seen in response to -adrenoceptor stimulation with isoprenaline. The maximal effect of phenylephrine (25% of the maximal effect of isoprenaline) required far higher concentrations (1 mmol/l) than isoprenaline (100 nmol/l); the EC₅₀ values amounted to 33.1 mol/l and 3.3 nmol/l, respectively. In the presence of the -adrenoceptor blocking agent phentolamine (1 mol/l), the concentration-response curve of phenylephrine was displaced to higher concentrations of the agonist; under these conditions, the EC₅₀ value amounted to 52.5 mol/l, The effects of the catecholamines noradrenaline and adrenaline on force of contraction remained unchanged in the presence of phentolamine (1 mol/l), or prazosin (1 mol/l), The positive inotropic effect of phenylephrine (1 mmol/l) was associated with a slight decrease in action potential duration; the effects on action potential were completely blocked in the presence of phentolamine (1 mol/l) These findings support the view that selective stimulation of -adrenoceptors may mediate a small but detectable positive inotropic effect in human atrial tissue under in vitro conditions. The requirement of high concentrations of -adrenoceptor agonists and the lack of effects of the endogenous catecholamines adrenaline and noradrenaline on -adrenoceptors (in concentrations which fully elicit the -adrenoceptors-mediated response) do not provide a basis for a functional role of -adrenoceptor-mediated effects under in vivo conditions. It is more likely that adrenaline- or noradrenaline-mediated changes in the force of contraction in the human atrium are virtually exclusively due to the stimulation of -adrenoceptors. Send offprint requests to H. Nawrath at the above address     

Alinidine reverses the descending staircase of isolated rat atria by an antimuscarinic action

Summary Contractile force of isolated atria from most mammalian species increases with the rate of electrical stimulation, resulting in an ascending staircase. In contrast, in the rat, contractile force decreases with increasing rate of stimulation (descending staircase). The bradycardic and antianginal drug alinidine (5.7–91.2 mol/l) reversed the descending staircase to ascending by a positive inotropic effect at higher stimulation rates. Maximal positive inotropy was obtained with 45.6 mol/l, a concentration which also caused maximal bradycardia in spontaneously beating atria. Concentrations of 1 mol/l of the antimuscarinic compounds atropine as well as the quarternary salt ipratropium bromide also reversed the descending staircase of rat atria. Addition of alinidine did not cause any further increase in force of contraction under these conditions. Addition of 1 mol/l physostigmine to isolated left atria from guinea pigs for blockade of acetylcholinesterase decreased contractility at all stimulation rates, but did not change the ascending character of the staircase. Alinidine antagonized the negative inotropic effect of physostigmine. The known antimuscarinic action of alinidine was quantified in electrically driven (0.25 Hz) left rat atria by antagonism of the negative inotropic effect of oxotremorine (0.01–10 mol/l). Alinidine acted as a strictly competitive antagonist with a pA₂ of 5.82. In isolated papillary muscle from guinea pigs, pretreated with reserpine for depletion of catecholamines, carbachol (0.1–3000 mol/l) exerted positive inotropic effects. Alinidine antagonized also this effect in a competitive fashion with a pA₂ value of 5.58. Investigations of the specific antimuscarinic compounds pirenzepine, methoctramine and AF-DX 116 in these models indicate that the negative inotropic effect in atria is mediated by M_2a-receptors while the positive inotropic effect in papillary muscle is mediated by either the M_2e-receptor or a yet unidentified muscarinic subreceptor.It is concluded that the descending staircase of electrically stimulated rat atria is due to the release of acetylcholine as well as to the short duration of its action potential which decreases further upon muscarinic stimulation, thus leading to a compromise of excitation-coupled calcium influx and negative inotropy, particularly at higher stimulation rates. Therefore, antimuscarinic drugs including alinidine reverse the descending staircase. Under physiological conditions of impulse generation and in the absence of vagal activity an ascending staircase is to be expected in rat atria as well.     

Muscarinic cholinoceptors in the human heart: demonstration,subclassification, and distribution

In human atrial and ventricular myocardium, the muscarinic cholinoceptor (M-cholinoceptor) populations were characterized by means of radioligand binding (with [N-methyl-3H]-scopolamine ([3H]-NMS) as the ligand) and functional experiments (negative inotropic effect of carbachol on isolated electrically driven right atrial and left papillary muscle preparations). (1) Binding of [3H]-NMS to human atrial and ventricular membranes was rapid, reversible and saturable (KD-values: 0.5-1.0 nmol/l). The maximal number of [3H]-NMS binding sites, however, was approximately 2.5-fold higher in right and left atrial membranes (200-250 fmol [3H]-NMS specifically bound/mg protein) than in right and left ventricular membranes (80-100 fmol/mg protein). (2) M-cholinoceptor antagonists inhibited [3H]-NMS binding to right atrial and left ventricular membranes with steep, monophasic competition curves indicating interaction with a single class of binding sites. In both tissues the order of potency was: atropine greater than AF-DX 116 greater than hexahydrosiladifenidol (HHSiD) greater than pirenzepine. (3) On isolated electrically driven right atrial and left papillary muscle preparations (with force of contraction enhanced by 10(-5) mol/l isoprenaline), carbachol (10(-8)-10(-4) mol/l) caused concentration-dependent decreases in force of contraction; the pD2-value for carbachol was 6.65 +/- 0.09 (n = 8, atria) and 6.62 +/- 0.08 (n = 10, papillary muscles). In both tissues M-cholinoceptor antagonists antagonized the negative inotropic effect of carbachol with an order of potency: atropine greater than AF-DX 116 greater than HHSiD greater than pirenzepine, identical to that obtained in radioligand binding experiments.(ABSTRACT TRUNCATED AT 250 WORDS)     

A neurally mediated inotropic effect of veratridine and cevadine on isolated guinea-pig papillary muscle

Summary The positive inotropic effect of veratridine and cevadine was investigated in the isolated, isometrically contracting guinea-pig papillary muscle. The increase of the force of the rested-state contraction, elicited after incubation of the resting muscle with veratridine or cevadine, served as a measure of the neurally mediated, sympathomimetic effect of these alkaloids. Concentrations exceeding 5 mol/l veratridine or 15 mol/l cevadine produced concentration-dependent increases of the force of the rested-state contraction. These concentrations are larger than those causing the maximum positive inotropic effect on muscles contracting continually at a rate of 1 Hz. The positive inotropic effect of 30 mol/l veratridine as manifested by the rested-state contraction was absent in the presence of 100 nmol/l tetrodotoxin and in muscles from reserpine-pretreated animals. It was significantly inhibited by 50 nmol/l (–)-propranolol, but not by the same concentration of (+)-propranolol. The effect of 30 or 60 mol/l cevadine was likewise absent in catecholamine-depleted preparations. It is concluded that the indirect inotropic effect of veratridine or cevadine, which is attributed to their noradrenaline-releasing effect on intracardiac nerves, requires higher concentrations than the direct positive inotropic effect, which is a consequence of the increased transsarcolemmal influx of Na ions into the myocardial cell.These results were communicated to the Deutsche Pharmakologische Gesellschaft (Honerjäger 1977)     

Evidence for adenosine receptor-mediated isoprenaline-antagonistic effects of the adenosine analogs PIA and NECA on force of contraction in guinea-pig atrial and ventricular cardiac preparations

Summary The effects of the adenosine agonists (–)-N⁶-phenylisopropyladenosine (PIA) and 5-N-ethylcarboxamideadenosine (NECA) on force of contraction, adenylate cyclase activity and normal as well as slow action potentials were studied in guinea-pig isolatedatrial (left auricles) andventricular preparations (papillary muscles).Inauricles PIA and NECA exerted concentration-dependent negative inotropic effects with similar potenticies (mean EC₅₀:0.05 mol l^–1 for PIA and 0.03 mol l^–1 for NECA). Similar results were obtained in the presence of isoprenaline.Inpapillary muscles PIA and NECA alone had no effect on force of contraction but produced negative inotropic effects in the presence of isoprenaline (mean EC₅₀:0.19 mol l^–1 for PIA and 0.10 mol l^–1 for NECA).In both preparations, the negative inotropic effects of PIA and NECA in the presence of isoprenaline were antagonized by the adenosine receptor antagonist 8-phenyltheophylline.In both preparations, PIA and NECA did not affect adenylate cyclase activity, both in the absence and presence of isoprenaline.Inauricles the negative inotropic effects of both nucleosides were accompanied by shortening of the action potential. This effect was also observed in the presence of isoprenaline. Inpapillary muscles the adenosine analogs did not detectably alter the shape of the normal action potential. Ca²⁺-dependent slow action potentials elicited in potassium-depolarized preparations also remained unaltered in the presence of PIA or NECA alone. However, the isoprenaline-induced enhancement of the maximal rate of depolarization of slow action potentials was attenuated by PIA or NECA.It is concluded that in guinea-pig atrial and ventricular cardiac preparations the adenosine analogs PIA and NECA exert isoprenaline-antagonistic effects on force of contraction via adenosine receptors the existence of which can thus be shown in a functional way. These receptors are not detectably coupled to the adenylate cyclase. The negative inotropic effect in theauricle is most likely due to a shortening of the action potential resulting from an activation of potassium channels, which in turn indirectly reduces the Ca²⁺ influx during the action potential. In theventricle the adenosine receptor is either not linked to these potassium channels or adenosine-sensitive potassium channels do not exist in the ventricle. Instead the activation of the receptor causes a decrease of the slow Ca²⁺ inward current but this effect is observed only when the slow Ca²⁺ inward current had previously been enhanced by a cyclic AMP-dependent mechanism.     

Muscarinic receptor interaction with full and partial β-adrenoceptor agonists in the rat colon strip

Summary -Adrenoceptor agonists inhibit contractile activity in isolated colon strips. In order to demonstrate that -adrenoceptors are located at different functional levels within the colon wall, increasing concentrations of muscarinic agonists were used to interact functionally with the -adrenoceptor-mediated inhibition of spontaneous colon activity. The effects of the full agonists isoprenaline and terbutaline and of the partial agonist prenalterol were functionally antagonized by carbachol (0.03 and 0.3 mol/l) and bethanechol (1,3 and 30 ol/l). This functional antagonism was parallelled by an increase in baseline tension and spontaneous contractile activity of the isolated colon strip. At lower concentrations of carbachol (0.003 and 0.01 ol/l) or bethanechol (0.03 and 0.31 mol/l) no effect on the contractile status of the smooth muscle or on the pD₂-values of the full agonists was seen. However, at these lower concentrations of muscarinic agonists a marked decrease in the maximal inhibitory response to the partial -adrenoceptor agonist prenalterol was demonstrated. The inhibitory response to prenalterol showed a biphasic concentration-response curve. The muscarinic antagonist atropine produced an increase in the maximal response of the high potency component of the concentration-response curve for prenalterol and an increase in the sensitivity to isoprenaline. These results demonstrate the presence of a high cholinergic tone in the colon preparation of a magnitude that clearly reduces the sensitivity to -adrenoceptor agonists. The different responses to full and partial -adrenoceptor agonists in the presence of increasing concentrations of muscarinic agonists may indicate that -adrenoceptors are located on two different functional units within the colon wall.     

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     

The effects of (−)-cromakalim and glibenclamide on uptake2 in guinea-pig trachealis muscle

Summary In a recent study, we have shown that hyperpolarization of cells by -adrenoceptor agonists results in stimulation of the uptake₂ process for catecholamines. The aim of the present study was to further explore the hypothesis that uptake₂ is dependent on membrane potential by examining the effects of the K⁺-channel opening drug, (–)-cromakalim, and the K⁺-channel blocking drug, glibenclamide, on uptake₂ of isoprenaline. The effects of these drugs were examined in guinea-pig trachealis muscle, in which isoprenaline and cromakalim cause hyperpolarization, and in rat heart, in which isoprenaline and cromakalim have little effect on membrane potential.In guinea-pig trachealis muscle segments, 1 mol/l glibenclamide reduced uptake₂ (as measured by the steady-state rate of corticosterone-sensitive formation of ³H-3-O-methylisoprenaline normalized for the isoprenaline concentration) in tissues incubated in concentrations of ³H-(±)-isoprenaline that hyperpolarize the muscle (25 and 250 nmol/l) but not at an isoprenaline concentration that did not hyperpolarize the muscle (1 nmol/l). (–)-Cromakalim (10 mol/l), which hyperpolarizes the trachealis muscle, increased uptake₂ of isoprenaline (1 or 25 nmol/l) and this effect of (–)-cromakalim was inhibited by glibenclamide. In rat hearts perfused with 1 or 25 nmol/l ³H-(±)-isoprenaline and 10 mol/l U-0521 to inhibit catechol-O-methyltransferase, the rate of uptake₂ of isoprenaline was unaffected by cromakalim or glibenclamide.The results show that hyperpolarization of cells by various mechanisms can result in stimulation of uptake₂ of catecholamines and provide further evidence to support the hypothesis that the uptake₂ transport process is driven by the membrane potential of cells.Preliminary results of part of this study were presented to the German Society for Pharmacology and Toxicology (Bryan-Lluka and Vuocolo 1991)Correspondence to L. J. Bryan-Lluka at the above address     

Mechanisms of the contractile effects of flosequinoxan

In guinea-pig papillary muscles the positive inotropic effect of flosequinoxan (BTS) starting at 100 mol/1 amounted to 287.6 ± 34.2% at 300 mol/l without any effects on time to peak tension (103.9 ± 2%) and relaxation time (107.1 ± 6.7% of predrug value, respectively). 10 mol/l carbachol attenuated the positive inotropic effect of 300 mol/l to 166.5 ± 11.6% (n = 10). The phosphorylation state of the inhibitory subunit of troponin (TnI) and phospholamban(PLB) in [³²P]-labeled guinea-pig ventricular myocytes was increased starting at 100 mol/l amounting to 142.5 ± 12.6% and 130.9 ± 2.2% at 300 mol/l, respectively (n = 5). Furthermore, BTS (300 mol/l) decreased phosphorylase phosphatase activity by 23.1%. It is concluded that the contractile effects of BTS are accompanied by enhanced phosphorylation of regulatory proteins which could in part be due to inhibition of phosphorylase phosphatase activity.     

Effects of isomazole on force of contraction and phosphodiesterase isoenzymes I-IV in nonfailing and failing human hearts.

The phosphodiesterase (PDE) inhibitor isomazole increased the force of contraction to 278.3 +/- 89.1% (n = 7) of the predrug value in ventricular trabeculae carneae isolated from nonfailing human hearts. This effect can be attributed mainly to a PDE III or a combined PDE III/IV inhibition since at the concentration of the maximal positive inotropic effect of isomazole, PDE III and PDE IV were completely inhibited. In explanted failing human hearts (end-stage myocardial failure, NYHA IV), isomazole increased the force of contraction only marginally to 110.1 +/- 10.7% of the predrug value. The lack of a distinct positive inotropic efficacy of isomazole in failing human hearts could not be explained by an impairment of PDE inhibition since the properties of the PDE I-IV isoenzymes separated by DEAE-Sepharose chromatography and the inhibitory effects of isomazole did not differ in both preparations. The positive inotropic effect of the beta-adrenoceptor agonist isoprenaline was also reduced in failing hearts. However, in the presence of isomazole, the diminished positive inotropic effect of isoprenaline was restored to values obtained with isoprenaline alone in nonfailing hearts. Thus, the decreased effect of inotropic drugs like isoprenaline or isomazole in preparations from failing human heart might be explained mainly by a diminished cAMP formation due to a defect in receptor-adenylate cyclase coupling.     

Effects of Ca2+ channel antagonists and ryanodine on H1-receptor mediated electromechanical response to histamine in guinea-pig left atria

Summary Effects of organic Ca²⁺ channel antagonists, Ni²⁺ and ryanodine on the electrophysiological and positive inotropic responses to histamine were examined in isolated guinea-pig left atria. Histamine increased force of contraction, prolonged action potential duration (APD) and hyperpolarized the membrane in a concentration-dependent manner. Histamine at a concentration of 1 mol/l produced a dual-component positive inotropic response composed of an initial increasing phase (initial component) and a second an late developing, greater positive inotropic phase (second component), whereas causing monophasic changes in APD and resting potential. The electrophysiological and dual-component positive inotropic effects induced by histamine were antagonized by chlorpheniramine (1 mol/l) but not by cimetidine (10 mol/l), indicating that both effects are exclusively mediated by H₁-receptors. The positive inotropic response to 1 mol/l histamine was changed by the pretreatment with nifedipine (1 mol/l) and nisoldipine (1 mol/l). In the presence of these dihydropyridines, the second component was almost completely abolished, while the initial component was hardly affected. On the other hand, verapamil (3 mol/l) and diltiazem (10 mol/l) failed to modify the multiphasic inotropic response to histamine. None of the Ca²⁺ channel antagonists affected the histamine-induced APD prolongation. In the presence of Ni²⁺ at a concentration of 0.3 mmol/l, at which it produced no negative inotropic action, the second component of the positive inotropic effect of histamine was specifically suppressed whereas the histamine-induced APD prolongation was unaffected. Preferential attenuation of the second component was also observed in the presence of 30 nmol/l ryanodine. However, the electrophysiological alterations caused by histamine remained unchanged. These results suggest that in guinea-pig left atria the H₁-receptor-mediated prolongation of APD seems unlikely to be due to enhancement of the slow inward Ca²⁺ current. Conversely, the increased Ca²⁺ influx as a result of the APD prolongation may contribute to the second component of the positive inotropic effect of histamine. Dihydropyridine Ca²⁺ channel antagonists, Ni²⁺ and ryanodine are all capable of inhibiting the second component, but do so possibly via different mechanisms, implying the complicated mechanisms underlying the H₁-receptor-mediated positive inotropic effect.Send offprint requests to Y. Hattori     

Nitric oxide influences the release of histamine and glutamate in the rat hypothalamus

To investigate the influence of nitric oxide (NO) on the release of histamine and glutamate, the anterior hypothalamus of anaesthetized rats was superfused through a push-pull cannula either with artificial cerebrospinal fluid (CSF) or with various drugs dissolved in CSF.Hypothalamic superfusion with the NO-donating compounds linsidomine (200 mol/l) or diethylamine-NO (DEANO, 100 mol/l) led to a pronounced and sustained decrease in the histamine release rate, whereas the release rate of glutamate was enhanced. Superfusion with the inhibitor of NO synthase L-N^G-nitro-L-arginine methyl ester (L-NAME, 200 mol/l) increased the histamine release rate. The inhibitory effect of 200 mol/l linsidomine was abolished by atropine (10 mol/l). Superfusion with the glutamate receptor agonists glutamate (100 mol/l) or N-methyl-D-aspartate (NMDA, 50 mol/l) enhanced the histamine release rate. In the presence of linsidomine, the releasing effect of NMDA was not changed.These findings demonstrate that the release of histamine in the hypothalamus is diminished by endogenous NO. This effect of NO on histamine release seems to be due to enhanced release of acetylcholine from vicinal cholinergic neurons via stimulation of muscarinic acetylcholine receptors located presynaptically on histaminergic neurons. The NO-induced glutamate release seems to exert a subordinate stimulatory effect on histamine release. Finally, the inhibition of histamine release by NO is not due to blockade of NMDA receptors.This work was supported by the Jubiläumsfonds der österreichischer Nationalbank     

Direct activation by okadaic acid of the contractile elements in the smooth muscle of guinea-pig taenia coli

Summary 1. Okadaic acid isolated from black sponge (Halichondria okadai), at the concentration of 10 mol/l, caused contraction in saponin-treated skinned smooth muscle of guinea-pig taenia coli in the absence of Ca²⁺. In the presence of low concentration (0.3 mol/l) of Ca²⁺ okadaic acid induced a greater contraction than in the absence of Ca²⁺ 2. Okadaic acid potentiated the contractions induced by Ca²⁺ and pCa²⁺-tension curve was shifted to the left as well as upward by 1 mol/l okadaic acid. 3. Native actomyosin preparation (myosin B) containing calmodulinmyosin light chain kinase system and phosphatase was obtained from taenia coli. Okadaic acid (10 mol/l) increased the actomyosin Mg²⁺-ATPase activity in the presence or absence of Ca²⁺. 4. Okadaic acid (1–100 mol/l) had no effect on calmodulin activity as monitored by Ca²⁺-calmodulin activated cyclic nucleotide phosphodiesterase activity and the (Ca²⁺ + Mg²⁺)-ATPase activity of erythrocyte membranes. 5. These results suggest that okadaic acid directly activates contractile elements of smooth muscle. Send offprint requests to H. Ozaki at the above address     

Suppression of isoprenaline-induced increase in plasma renin concentration by vasoconstrictors in rats with nonfunctioning Macula densa

Summary The mechanism of the increase in plasma renin concentration caused by the -sympathomimetic agent isoprenaline has been further investigated.Rats were pretreated by occluding the left renal artery for 2 hrs, thus rendering the macula densa cells of this kidney nonfunctioning. After contralateral nephrectomy infusion of isoprenaline (1.5 g/kg min) still caused a strong increase in plasma renin concentration. This increase was significantly suppressed by simultaneous infusion of angiotensin II (1.0 g/kg min), the -sympathomimetic amine phenylephrine (60 g/kg min) or octapressin (10 mU/kg min). The results exclude any mediator-role of the macula densa receptors in the isoprenaline-induced release of renin.The possibility of a stimulation of renin release via the baroreceptors or a direct secretomotoric action of isoprenaline is discussed.Supported by Deutsche Forschungsgemeinschaft Me 541/1.