Safety of cyproterone acetate: report of active surveillance.

Concern about a potentially increased risk of liver cancer associated to cyproterone acetate (CPA) treatment led to a postmarketing long-term surveillance study with historic accural of four groups of patients with sexual-hormonal disorders under treatment with CPA. The aim of the study was to provide a description of potential ADRs under CPA treatment with special emphasis on liver cancer. A long-term follow-up of 2506 patients was conducted. Six hundred and two persons were followed up retrospectively for longer than 10 years. In 16,721 patient-years of observation after the first CPA dose no malignant liver tumour was observed, whereas six would have been expected in this cohort. Seven incident, non-fatal benign liver tumours were found. Altogether 9.6% of a subset of 1685 patients with reported liver tests had, at some time, elevated liver enzymes. No cases were reported where CPA therapy had been discontinued due to severe liver disorder. We concluded from this active surveillance project, that CPA treatment is probably safe in the groups of patients followed up. Even though there was not a single case of liver cancer detected, the hypothesis of an elevated risk is being tested in an ongoing collaborative European case-control study which is examining the association of CPA use and primary hepatocellular cancer.

4-(4-Guanidinobenzoyl)-2-imidazolones and related compounds: phosphodiesterase inhibitors and novel cardiotonics with combined histamine H2 receptor agonist and PDE III inhibitor activity.

A series of new positive inotropic agents was synthesized with the aim of combining the pharmacophores of the imidazolone-type phosphodiesterase (PDE) inhibitor enoximone and guanidine-type histamine H2 receptor agonists such as arpromidine. All compounds are para-substituted 4-benzoyl-5-alkyl-2-imidazolones. H2 agonism was incorporated by p-(hetero)arylalkyl substituents, in particular by an imidazolylpropyl guanidine group. In addition analogous ureas, cyanoguanidines, alkyl guanidine carboxylates, and amides were prepared. These functional groups were either directly attached to the phenyl ring or linked by an appropriate spacer. The compounds were screened for positive inotropic activity in the isolated electrically stimulated guinea pig papillary muscle and for inhibition of PDE III (cGMP-inhibited cAMP PDE, isolated from guinea pig heart). The cardiotonics obtained proved to be either PDE III inhibitors, some of them surmounting up to 3-fold the potency of enoximone, or pharmacological hybrids combining both PDE III inhibitor and histamine H2 receptor agonist activities. These hybrids were the most potent positive inotropic substances at the papillary muscle, probably due to their synergistic mechanism of action. The participation of histamine H2 receptors could be demonstrated in the papillary muscle preparation by pretreatment with the H2 antagonist famotidine (10 microM) as well as by further pharmacological experiments using isolated perfused hearts of guinea pigs and rats, isolated guinea pig right atria, adenylyl cyclase and H2 receptor binding assays. At equieffective concentrations the moderate PDE III inhibitor and histamine H2 agonist N1-(4-[(1,3-dihydro-5-methyl-2-oxo-3H-imidazol-4-yl)-carbonyl]phenyl)-N2 - [3-(1H-imidazol-4-yl)propyl]guanidine 65 and the 5-ethyl homologue 66 were about 2 and 10 times more potent than enoximone at the papillary muscle. Moreover, both compounds produced a 2.5-fold higher maximal response than the reference compound.

Immunocytochemical studies of the Gi protein mediated muscarinic receptor-adenylyl cyclase system.

The localization of three key signal transduction components was indicated in rat heart tissue by immunocytochemical and histochemical experiment. It was shown that: 1. The M2 muscarinic receptors are localized along outer cell membranes and T-tubule membranes of cardiomyocytes but additionally at membranes of endothelial cells and fibroblasts. 2. Gia was found along outer cell membranes of cardiomyocytes and other cells of the heart and also inside the cells of the perinuclear space in close contact to the nuclei envelope and the endoplasmic reticulum membranes. Goa were found to be associated mainly in atrial tissue, especially at the nerval (neuronal) endings located among the cardiac muscle cells. This was shown in parallel incubation with specific neuronal antibody as a marker for these structures. 3. Adenylyl cyclase was localized along the sarcolemma and the T-tubule membranes in normal cardiomyocytes of rat and guinea pig hearts. Under ischemic conditions, the adenylyl cyclase was also seen in junctional sarcoplasmic reticulum membranes. The reasons for this changed localization need further elucidation. Binding of the adenylyl cyclase within the molecular structure of the membrane or variation of the marker penetration remain to be clarified.

Pituitary adenylate cyclase activating peptides are endothelium-independent dilators of human and porcine coronary arteries.

The PACAPs have been shown to be potent vasodilators in different animal species. Data in humans are still lacking. Therefore we investigated the effects of PACAP 38, PACAP 27 and VIP on isolated human and porcine coronary arteries (HCA and PCA). Our data show, that the PACAPs are endothelium-independent vasorelaxants, which in HCA are slightly more potent than VIP. The N-terminal shortened peptides PACAP 6-38 and PACAP 6-27 also show relatively potent vasorelaxant effects, acting as partial agonists. Glibenclamide, a selective inhibitor of ATP-sensitive potassium channels, partially reverses the effects of the PACAPs, indicating an involvement of these channels in the mechanism of action.

Comparative immunocytochemical demonstration of G proteins in rat heart tissue.

Localization of G proteins in the rat heart tissue was investigated using primary affinity-purified antibodies against synthetic peptides with amino acid sequences corresponding to alpha-subunits (alpha i common and alpha i 1, 2) of G proteins. Detection of immunoreactivity was performed with the peroxidase-anti-peroxidase complex (PAP), avidin-biotin complex (ABC) and fluorescein-labelled secondary antibodies for light microscopy and the protein A-gold technique for electron microscopy. In ventricles and atria, immunostaining for G proteins was detected in the sarcolemma and perinuclear space of cardiomyocytes. In endotheliocytes and fibroblasts, immunoreactivity was present also in the endoplasmic reticulum. All four immunocytochemical methods permit to demonstrate the same localization of G proteins in heart tissue. The ABC method and fluorescein labelled secondary antibodies technique showed the same sensitivity which is higher than that of the PAP method. Nomarski contrast microscopy enhanced the visualization of the final reaction product formed by the peroxidase reaction developed with diaminobenzidine in the ABC method. The results are discussed in terms of the role of G proteins in signal transduction via plasma membrane and membranes of the intracysternal space of the cell.

Diltiazem prevents the depression of adenylyl cyclase activity induced by the calcium paradox in rat.

In isolated rat hearts the calcium paradox, induced by perfusion for 3 minutes in the absence of calcium followed by perfusion for 10 minutes in the presence of calcium, depressed the activation of adenylyl cyclase by l-isoproterenol, NaF and forskolin. The characteristics of the beta-adrenoceptors and the activation of adenylyl cyclase by guanylyl imidodiphosphate were not changed. The findings suggest an uncoupling of beta-adrenoceptors from the catalytic site of the adenylate cyclase complex. Diltiazem, at 0.4 microM in the perfusion medium, greatly reduced the diminution of the activation of adenylate cyclase by isoproterenol and forskolin, and completely prevented the depression of the activation of adenylate cyclase by NaF. These effects may be due to interference by diltiazem with the mechanisms that promote an excessive influx of calcium into the heart during the calcium paradox.

[Clinical and hemodynamic results of surgical correction of hypertrophic obstructive cardiomyopathy]. / Klinische und hämodynamische Ergebnisse nach operativer Korrektur bei hypertroph obstruktiver Kardiomyopathie (HOCM).

The value of transaortal subvalvular myectomy after Morrow remains unclarified. We therefore analysed our results with HOCM with particular attention to the operation risk and the longterm results. 56 patients were treated at the Leipzig Heart Centre between January 1984 and August 1990 using the transaortic myectomy. In 16 patients an additional mitral valve replacement or -reconstruction was required. In 14 patients, other combined operations (aortic valve replacement, aortocoronary bypass) were indicated. In the postoperative observation period (up to 7 years; 141 patient-years; mean follow-up 4.2 yrs) detailed information was obtained at regular intervals about subjective complaints, ECG changes, left ventricular functional parameters and the weight of the heart muscle mass were recorded. The myectomy resulted in an alteration of the NYHA-class from 3.1 to 1.3 postoperatively (p < 0.05). The ventriculo-aortal pressure gradient reduced in the group myectomy (group I) from 69.2 +/- 5.2 to 23.3 +/- 2.7 mmHg postoperatively. In the myectomy+mitral valve repair group (group II) the intracavitary pressure gradient was even reduced to 11.7 +/- 2.2 Torr (p < 0.05). The Sokolov-Lyon-Index was 3.7 +/- 0.19 mV preoperatively and went down to 2.9 +/- 0.16 mV. The heart muscle mass decreased from 680 g to a postoperative value of 430 g (p < 0.05). The relation of BAR and calcium channel density of 0.5 +/- 0.1 in HOCM versus 0.9 +/- 0.08 in a control group (n = 6) proves the increased number of calcium channels in HOCM.

Comparison of myectomy alone or in combination with mitral valve repair for hypertrophic obstructive cardiomyopathy.

UNLABELLED: Between 1/84 and 6/91 56 patients were treated for hypertrophic obstructive cardiomyopathy (HOCM): the Morrow technique alone was performed on 40 patients (group 1), in 16 patients (group 2) an additional replacement (n = 13) or reconstruction (n = 3) of the mitral valve was indicated. In a total of 14 cases coronary artery bypass grafting and aortic valve replacement was performed in addition. Postoperatively (mean follow-up 4.2 yrs, 141 patient-years) left-ventricular diastolic and systolic function parameters, heart muscle mass, ECG findings, and symptomatology were recorded and the ratios of beta-adrenoreceptor density to density of the calcium channel were measured. RESULTS: Pressure gradient decreased from 69.2 +/- 5.2 (group 1) and 75.1 +/- 4.8 (group 2) to 23.3 +/- 2.7 and 11.7 +/- 2.2 mmHg postoperatively. Likewise Sokolow-Lyon index decreased from 3.5 +/- 0.2/3.7 +/- 0.2 to 2.9 +/- 0.2/2.8 +/- 0.3. The quotient time-to-peak-velocity/left-ventricular-ejection-time decreased significantly in group 2 from 58.6 +/- 6.3 to 41.9 +/- 5.8 (p less than 0.05). The heart muscle mass, determined echocardiographically, decreased from 680g to 430g (p less than 0.05). Isovolumetric tension time, isovolumetric relaxation time, and E/A ratio at rest and after stress showed typical characteristics. Ca(++)-channel density was clearly raised in all patients, with no differences between the two groups being observable. We conclude from our results: The most marked improvements in clinical and left-ventricular functional parameters were experienced by patients in group 2 (myectomy+MVR).(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro effects of reactive O2 species on the beta-receptor-adenylyl cyclase system.

The irreversible loss of activity of the sarcolemma-localized beta-receptor-adenylyl cyclase system (beta-RAS) in myocardial ischemia is a well documented phenomenon. Alterations in the sarcolemma (SL) induced by reactive O2 species could be responsible for this loss. Therefore the influence of oxidation of SH-groups and lipid peroxidation induced by Fe2+/Vit. C on the beta-RAS activity was studied. During incubation of SL with Fe2+/Vit. C a transient enhancement followed by a continuous loss of the beta-RAS activity (isoprenaline-, NaF-, Gpp(NH)p-, forskolin-stimulated and basal activity) was observed. In contrast there occurred a continuous loss of SH-groups and lipid peroxidation, beginning immediately after the start of incubation. Loss of SH-groups and lipid peroxidation as well as changes in the beta-RAS did not take place in the presence of the antioxidant t-Butyl-4-hydroxyanisole (BHA) or the Fe(2+)-chelator EGTA. In view of the known ischemia-induced formation of reactive O2 species our results show that these powerful oxidants could contribute to the modulation of the beta-RAS during myocardial ischemia.

Immunocytochemical localization of G-proteins (alpha subunits) in rat heart tissue.

The subcellular localization of peptide antibodies against G-protein alpha subunits was studied by the indirect immunogold technique with Lowicryl K4M embedded rat cardiac tissue. Two antibodies were used. The alpha common peptide antibody recognizes the alpha subunits of Gs, Gi and Go. The alpha i common peptide antibodies recognize the alpha subunits of all Gi alpha subtypes (G1-3). Immunoreactivity against alpha common and alpha i common antibodies was found along the cell surface of cardiomyocytes and endothelial cells. No immunoreactivity was seen on sarcoplasmic reticulum membranes. T-tubule membranes showed a little reactivity. Distribution patterns obtained with alpha common and alpha i common antibodies were identical. Immunogold was seen in cardiocytes from both atria and ventricles. In light-microscopical studies with peroxidase-conjugated secondary antibodies, heavy immunostaining was seen in SA and AV nodes.

G protein function in the ischaemic myocardium.

The activity of adenylyl cyclase (AC) is controlled by its interaction with receptor-regulated G proteins. The efficiency to form cyclic AMP is strongly influenced by the amount, the subspecies and function of these regulatory proteins. An impairment of AC function has been shown to occur in sarcolemmal preparations (SL) of hearts exposed to either local or global ischaemia. To examine the contribution of G protein function to this phenomenon, cholera toxin (CT)-catalysed ADP-ribosylation of Gs and pertussis toxin (PT)-catalysed ADP-ribosylation of G proteins have been investigated in SL of porcine hearts exposed to global ischaemia for 15-45 min. ADP-ribosylation by CT of an approximately 45 kDa polypeptide was 0.46 +/- 0.06 and ADP-ribosylation by PT of three 39-41 kDa polypeptides was 4.77 +/- 0.77 pmol mg-1 protein in SL of non-ischaemic myocardium. Whereas no change was observed in CT-catalyzed ribosylation after 30 min of ischaemia, there was a reduction in PT-catalyzed ADP-ribosylation to 3.7 +/- 0.35 pmol mg-1 protein after 30 min of ischaemia. Prolongation of ischaemia to 45 min did not reduce further ADP-ribosylation capacity. Quantitative immunoblotting of PT-sensitive G proteins suggests that the diminution of ADP-ribosylation occurred because of a loss of alpha-subunits of G0, Gi-1, and Gi-2 from sarcolemmal membranes.

Free radical-induced damage of cardiac sarcolemma (SL) and activity loss of beta-receptor adenylate cyclase system (beta-RAS). A comparison of the time courses.

In vitro studies have demonstrated that free radicals generated by Fe2+/Vit. C alter the beta-RAS function. SH-oxidation, peroxidation of sarcolemmal lipids and reaction of aldehydes (formed by lipid peroxidation) with the beta-RAS may contribute to this effect. In order to clarify the role of these reactions in respect to their ability to alter the beta-RAS function the time course of SH-oxidation, lipid peroxidation and formation of aldehyde reaction products has been studied. As result it is shown that SH-oxidation is nearly completed within 5 minutes and lipid peroxidation within 30 minutes after exposure to Fe2+/Vit. C. During this time period there was only a very small amount of aldehyde reaction products detectable. Therefore SH-oxidation and/or lipid peroxidation should be responsible for the activity loss of the beta-RAS.

Investigations on the mechanism of a diminished inotropic isoprenaline effect in isolated rat hearts after high linoleic acid diet.

Male Wistar rats were fed on a diet rich (13.3 J%) or poor (0.5 J%) in linoleic acid for 10 weeks. The inotropic and chronotropic effects of isoprenaline (100 ng) in isolated perfused hearts were reduced after a linoleic acid rich diet by 36% and 51%, respectively, with an accompanying reduction in isoprenaline-induced increase in the ventricular cAMP content. Moreover, the stimulation of adenylate cyclase in homogenates of ventricular tissue was diminished after linoleic acid rich diet. We postulate that the diminished adrenergic cardiac response after high vs. low linoleic acid diets could be due to alterations in the adenylate cyclase activity and cAMP formation.

Altered effect of BAY K 8644 after exposure of the rat aorta to IBMX or Db cAMP.

The development of isometric tension of aortic rings from rats was tested after cumulative administration of BAY K 8644 before and after exposure of the aortic preparations for 15 or 60 minutes to IBMX (10(-4) M) or Db cAMP (3.10(-4) M). BAY K 8644 exhibits dose-dependent contractions of those aortic rings which have not been exposed or have been exposed for only 15 minutes to IBMX or Db cAMP. BAY K 8644 application, after an exposure time of 60 minutes of the aortic rings to either IBMX or Db cAMP, resulted in a dose-dependent relaxation. This relaxing effect is counteracted by elevation of KCl (15 mM) in bath solution but was not changed by norepinephrine. The results indicate that long time exposure of the aortic rings to compounds capable of elevating intracellular cAMP level might induce changes of Ca channel gating.

Decreased contractile response after exposure of the rat aorta to Db cAMP or IBMX.

Isometric tension developed by different receptor agonists was found to be decreased after pretreatment of rat aortic rings with IBMX or Db cAMP and only partially restored by CaCl2 and A 23187. The contraction produced by Bay k 8644 was converted into dose-dependent relaxation after pretreatment of the aorta rings with Db cAMP or IBMX. An alteration of the calcium channel is assumed to play a common role in the cAMP-dependent inhibition of the smooth muscle contraction.

Responsiveness of cardiac adenylate cyclase in the normal and ischemic myocardium. Role of oxygen free radicals.

Cyclic AMP has been shown to play a significant regulatory role in a number of myocardial cell functions. The cAMP-forming adenylate cyclase complex is localized in the sarcolemmal membrane which is the major site of lipid peroxidation by oxygen-derived free radicals known to be increased in the ischemic myocardium. Adenylate cyclase function was found to be depressed in the ischemic myocardium but the specific biochemical mechanism responsible for this effect is still unknown. Therefore, the effect of free radical formation on adenylate cyclase was studied. Highly purified sarcolemmal membranes, exhibiting an NaF-stimulated activity of 3.46 +/- 0.65 nmol cAMP formed min-1 mg-1 of protein, were exposed to free radicals formation of which was induced by Fe++/ascorbate. A rapid loss of adenylate cyclase activation by 1-isoproterenol, NaF, and Gpp(NH)p has been observed. Concentration of malondialdehyde (MAD), a key intermediate in the formation of peroxides, was positively correlated to the rapid loss of adenylate cyclase activity. As result, adenylate cyclase was found to be highly susceptible to free radical-induced damage. It is thought that this effect might be one of the causes of the biochemical dearrangements contributing to the alteration of sarcolemmal membrane function in myocardial ischemia resulting in the reduction or loss of metabolic and contractile regulation of the heart.

Putative Ca2+ channels in cardiac membranes. Subcellular distribution of [3H]nitrendipine receptors.

The binding of Ca2+ channel blocking drug nitrendipine was studied in purified sarcolemma (SL), fragmented sarcoplasmic reticulum (SR), and crude membranes isolated from porcine left ventricle. The density of specific [3H]nitrendipine binding sites was compared to Na+/Ca2+ exchange and (Na+, K+)ATPase activities of each membrane preparation. Enrichment of [3H]nitrendipine binding sites in purified SL correlates excellently with the purification of the two studied sarcolemmal marker activities. The results suggest that high affinity nitrendipine receptors are solely localized in cardiac SL.

Adrenergic regulation of the acute ischaemic myocardium: facts, interpretations and consequences.

Anoxic stress is accompanied by activation of the central and peripheral sympathetic nervous system resulting in a high local catecholamine concentration. The authors studied how myocardial cells cope with the high level of catecholamines under ischaemic conditions. The beta-adrenoceptor-adenylate cyclase system (AC) was investigated in different models of ischaemia and anoxia (global ischaemia, low-perfused hearts, coronary artery ligation) in rat hearts. It was shown that beta-receptor function is not changed up to 40 min of ischaemia. Myocardial AC function was depressed in the total ischaemic myocardium but not in the low-perfused hearts indicating a non-uniform alteration of AC function. Reduced AC activity was completely reversible by aerobic perfusion as long as the ischaemic period did not exceed 20 min. Depression of AC function during severe ischaemia was avoided by reducing Ca2+ in the extracellular fluid and by pretreatment with Ca2+ channel blockers (verapamil). Depression of AC function during severe ischaemia is caused mainly by increased intracellular Ca2+ which inhibits AC at its catalytic site. Myocardial ischaemia alters the response of myocardial cells to catecholamines and other activators of the AC system. This alteration is time-limited and turns damage to AC function from reversible to irreversible after prolongation of ischaemia to more than 30 min.

Signal transfer in cardiac muscle. Alteration of the beta-adrenoceptor adenylate cyclase system in the hypertrophied myocardium.

The beta-adrenoceptor adenylate cyclase complex (beta ACR), located in the sarcolemmal membrane, is one of the most effective signal transduction systems regulating function and metabolism of heart muscle primarily via cyclic AMP. It is thought to play an important role in adaptive mechanisms of the heart as to pressure load and stressful stimuli. Present knowledge about composition and function of beta ARC enable us to clear up which of the single components of this system contributes to pathophysiological alterations of heart function. Cardiac beta ARC was investigated in three experimental groups: I) adult rats of WKY strain (WKY), II) adult rats of WKY strain cardiac hypertrophy in which was induced by aortic constriction (WKYAC), and III) adult spontaneously hypertensive rats (SHR). Quantitative assessment of beta-adrenoceptor number (beta AR) as revealed by [3H]dihydroalprenolol binding studies showed a significant reduction to 30% and 35% of control beta AR in membrane preparations of WKYAC and SHR, respectively. The diminished density of myocardial beta AR was accompanied by a reduction of the maximum stimulatory effect of 1(-)adrenaline on adenylate cyclase (AC). Evidence was obtained for a close correlation between the diminished response of beta ARC to beta AR-mediated stimuli and the heart index as measure of cardiac hypertrophy. No correlation between graduated states of hypertrophy and activation of AC has been observed by NaF and Gpp(NH)p. The results indicate that in rat hearts severe hypertrophy of which was induced by pressure-load, mainly the beta AR component of the beta ARC complex contributes to the reduction of beta ARC-mediated responsiveness of the hypertrophied myocardium.