Regional expression of phospholamban in the human heart.

BACKGROUND: Several independent lines of evidence indicate that phospholamban (PLB) expression correlates positively with depression of force of contraction and duration of contraction in isolated cardiac preparations of several animal species. Here, we studied whether PLB levels correlate with attenuation of contractility and enhancement of contractile time parameters in different parts of the human heart. METHODS: Force of contraction was measured in isolated electrically driven atrial and ventricular preparations from human hearts. Ca(2+)-uptake by human atrial and ventricular homogenates was assayed at different ionized Ca(2+)-concentrations. Protein expression of PLB and the sarcoplasmic Ca(2+)-ATPase (SERCA) was measured in homogenates by quantitative immunoblotting using specific antibodies. PLB mRNA expression was quantified in human cardiac preparations by Northern blot analysis. RESULTS: The duration of contraction in isolated preparations of human right ventricle (RV) was double that found in right atrial preparations (RA) (620 +/- 25 ms versus 308 +/- 15 ms). In RA, PLB expression was reduced by 44% at the protein level and by 34% at the mRNA level compared to RV. In contrast, the SERCA protein content was increased by 104% in RA compared to RV. Ca(2+)-uptake at low ionized Ca(2+)-concentration, where the inhibiting effect of PLB is maximal, amounted to 1.39 +/- 0.28 nmol Ca2+/mg protein in RA and to 0.62 +/- 0.09 nmol Ca2+/mg protein in RV (n = 6 both). CONCLUSIONS: It is suggested that duration of contraction is shorter in human atrium versus ventricle due to the combined effect of decreased PLB levels (which inhibits SERCA function) and increased SERCA levels. The lower relative ratio of PLB to SERCA leads to less inhibition of SERCA and increased Ca(2+)-uptake which enhances relaxation and contraction in human atrium.

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.

The mechanism of action of cantharidin in smooth muscle.

1. The aim of this study was to investigate the mechanism(s) of the vasoconstrictor effect of cantharidin in bovine preparations. 2. Catalytic subunits of protein phosphatase type 1 (PP 1) and type 2A (PP 2A) were immunologically identified in coronary arteries, isolated smooth muscle cells and ventricular myocardium. 3. The mRNAs coding for catalytic subunits of PP 1alpha, PP 1beta and PP 2Aalpha were identified by hybridization with specific cDNA-probes in total RNA from coronary arteries, isolated smooth muscle cells and ventricles. 4. The activities of catalytic subunits of PP 1 and PP 2A separated by column chromatography from coronary arteries, isolated smooth muscle cells and ventricles were inhibited by cantharidin in a concentration-dependent manner. 5. Cantharidin increased the phosphorylation state of smooth muscle proteins including the regulatory light chains of myosin in 32P-labelled intact smooth muscle cells in a concentration-dependent manner. 6. Cantharidin did not affect cytosolic calcium concentrations in aortic smooth muscle cells. 7. It is suggested that cantharidin contracts smooth muscle preparations by increasing the phosphorylation state of regulatory proteins due to inhibition of phosphatase activities. Thus, cantharidin might be a useful tool to study the function of phosphatases in smooth muscle.

The effect of the protein phosphatases inhibitor cantharidin on beta-adrenoceptor-mediated vasorelaxation.

1. Cantharidin, an inhibitor of protein phosphatase types 1 (PP1) and 2A (PP2A), increased basal tone of bovine isolated coronary artery rings (CARs) with and without endothelium in a time- and concentration-dependent manner with pEC50 values of about 5.1 and 5.2, respectively, for both preparations. 2. Beta-Adrenoceptor stimulation with isoprenaline (Iso; 0.03-100 microM) or inhibition of phosphodiesterase activity by 3-isobutyl-1-methylxanthine (IBMX; 10-1000 microM), respectively, relaxed CARs precontracted with KCl (75 mM). CARs with and without endothelium showed no difference in the relaxing response to Iso and IBMX, respectively. 3. Cantharidin (3 microM) attenuated vasorelaxation induced by Iso (0.03-100 microM) in CARs with and without endothelium in a time-dependent manner, whereas vasorelaxation induced by IBMX (10-1000 microM) was not attenuated by 3 microM cantharidin. 4. Cantharidin (3 microM) did not affect cyclic AMP content in bovine cultured vascular cells, i.e. coronary artery smooth muscle cells (BCs), aortic endothelial cells (BAECs) and aortic smooth muscle cells (BASMCs), either under basal conditions, after beta-adrenoceptor stimulation (Iso) or inhibition of phosphodiesterase activity (IBMX), respectively. 5. Cantharidin inhibited protein phosphatase activity in homogenates from bovine coronary artery rings with a pIC50 of about 6.0. In homogenates of bovine cultured vascular cells pIC50 values of cantharidin amounted to about 6.5 for BCs, 6.7 for BAECs and 6.7 for BASMCs, respectively. 6. It was concluded that cantharidin differently affects vasorelaxation due to stimulation of beta-adrenoceptors (Iso) or inhibition of phosphodiesterase activity (IBMX), respectively. The attenuation of beta-adrenoceptor-mediated vasorelaxation by phosphatase inhibition is not due to diminished adenosine 3':5'-cyclic monophosphate (cyclic AMP) generation but could be evidence for different subcellular compartments of cyclic AMP.

Functional studies in atrium overexpressing A1-adenosine receptors.

1. Adenosine and the A1-adenosine receptor agonist R-PIA, exerted a negative inotropic effect in isolated, electrically driven left atria of wild-type mice. 2. In left atria of mice overexpressing the A1-adenosine receptor, adenosine and R-PIA exerted a positive inotropic effect. 3. The positive inotropic effect of adenosine and R-PIA in transgenic atria could be blocked by the A1-adenosine receptor antagonist DPCPX. 4. In the presence of isoprenaline, adenosine exerted a negative inotropic effect in wild-type atria but a positive inotropic effect in atria from A1-adenosine receptor overexpressing mice. 5. The rate of beating in right atria was lower in mice overexpressing A1-adenosine receptors compared with wild-type. 6. Adenosine exerted comparable negative chronotropic effects in right atria from both A1-adenosine receptor overexpressing and wild-type mice. 7. A1-adenosine receptor overexpression in the mouse heart can reverse the inotropic but not the chronotropic effects of adenosine, implying different receptor-effector coupling mechanisms.

Negative chronotropic and inotropic effects exerted by diadenosine hexaphosphate (AP6A) via A1-adenosine receptors.

1. Diadenosine hexaphosphate (AP6A) exerts vasoconstrictive effects. The purpose of this study was to investigate whether AP6A has any effect on cardiac function. 2. The effects of AP6A (0.1-100 microM) on cardiac contractility and frequency were studied in guinea-pig and human isolated cardiac preparations. Furthermore, the effects of AP6A on the amplitude of the L-type calcium current, on the adenosine 3':5'-cyclic monophosphate (cyclic AMP) content and on the phosphorylation of regulatory phosphoproteins, i.e. phospholamban and troponin inhibitor, were investigated in guinea-pig isolated ventricular myocytes. 3. In isolated spontaneously beating right atria of the guinea-pig AP6A exerted a negative chronotropic effect and reduced the rate of contraction maximally by 35% (IC20 = 35 microM). 4. In isolated electrically driven left atria of the guinea-pig AP6A exerted a negative inotropic effect and reduced force of contraction maximally by 23% (IC20 = 70 microM). 5. In isolated electrically driven papillary muscles of the guinea-pig AP6A alone was ineffective, but attenuated isoprenaline-stimulated force of contraction maximally by 23% (IC20 = 60 microM). Furthermore, AP6A attenuated the relaxant effect of isoprenaline. 6. In human isolated electrically driven ventricular preparations AP6A alone was ineffective, but attenuated isoprenaline-stimulated force of contraction by maximally 42% (IC20 = 18 microM). Moreover, AP6A attenuated the relaxant effect of isoprenaline. 7. All these effects of AP6A were abolished by the selective A1-adenosine receptor antagonist 1,3-dipropyl-cyclopentyl-xanthine (DPCPX, 0.3 microM), whereas the M-cholinoceptor antagonist atropine (10 microM) and the P2-purinoceptor antagonist suramin (300 microM) failed to abolish the effects of AP6A. 8. AP6A 100 microM had no effect on the amplitude of the L-type calcium current, but attenuated isoprenaline-stimulated L-type calcium current. The maximum of the current-voltage relationship (I-V curve) was shifted to the left by isoprenaline and additional application of AP6A shifted the I-V curve back to the right to the control value. The phosphorylation state of phospholamban and the troponin inhibitor was unchanged by AP6A alone, but was markedly attenuated by AP6A in the presence of isoprenaline. Cyclic AMP levels remained unchanged by AP6A, even after stimulation with isoprenaline. 9. In summary, AP6A exerts negative chronotropic and inotropic effects in guinea-pig and human cardiac preparations. These effects are mediated via A1-adenosine receptors as all effects were sensitive to the selective A1-adenosine receptor antagonist DPCPX. Furthermore, the effects of AP6A on cyclic AMP levels, protein phosphorylation and the L-type calcium current are in accordance with stimulation of A1-adenosine receptors.

Inositol-1,4,5-trisphosphate increase by diadenosine tetraphosphate in preparations from failing human myocardium.

In human ventricular trabeculae carneae 100 microM AP4A (diadenosine tetraphosphate) increased force of contraction to 162.8+/-15.7% of predrug value (n=9). This positive inotropic effect was accompanied by a prolongation of time parameters: time to peak tension and time of relaxation were prolonged by 7.8+/-1.3% and 14.9+/-3.8%, respectively (P<0.05). In the same trabeculae, AP4A increased IP3 (inositol-1,4,5-trisphosphate) content from 9.0+/-1.3 pmol/mg to 22.9+/-5.4 pmol/mg protein (n=5-9). In conclusion, the positive inotropic effect of AP4A in the human myocardium is likely due to an increase of IP3 mediated probably via Gq-coupled P2Y-purinoceptors. Because of the prominent role of Gq in the development of cardiac disease, these findings may lay the ground to further investigate the possible role of AP4A and/or related ligands (e.g. AP2A and AP3A) in heart failure.

On the contractile function of protein phosphatases in isolated human coronary arteries.

It is unknown whether protein phosphatases types 1 and 2A are present in and can regulate the tone of human vascular tissue. The expression and possible function of serine/threonine protein phosphatases (PP) type 1 (PP1) and type 2A (PP2A) were studied in isolated human coronary arteries. Catalytic subunits of PPI and PP2A were identified by means of phosphatase activity measurement in tissue homogenates, by separation of enriched extracts through affinity column chromatography, by immunoblotting with specific antibodies, by hybridization of mRNA with specific DNA probes and PCR of reverse transcribed mRNA. Based on these methods, the catalytic subunits of PP1(alpha,beta,gamma) and PP2A(alpha,beta) were identified. Appropriately, cantharidin, an inhibitor of PP1 and PP2A, increased basal tone of human isolated coronary artery rings with an EC50 of about 16 micromol/l by increasing the phosphorylation state of the regulatory light chains of myosin. In summary, PP1 and PP2A are expressed in human coronary arteries and they can alter vascular tone.

Analysis of the components of Lycopus europaeus L. in body fluids during metabolism studies. Comparison of capillary electrophoresis and high-performance liquid chromatography.

During pharmacokinetic studies with extracts obtained from medicinally used plants, analysis in body fluids is mainly performed by HPLC, an established separation method. In this paper high-performance capillary electrophoresis (HPCE) is investigated for its ability to separate such complex extracts. Crude extracts of Lycopus europaeus L. (Lamiaceae) are traditionally used against mild forms of hyperthyroidism. The metabolism of a 70% ethanolic extract with respect to some of its individual main components (rosmarinic and caffeic acid, luteolin-7-glucoside) and a mixture of the pure compounds were investigated using isolated perfused rat liver. After solid-phase extraction metabolites were determined using HPCE and HPLC separation techniques. A buffer solution composed of 0.05 mol l-1 Na2HPO4 at pH 7.0 with 30% acetonitrile was found to be the most suitable electrolyte for HPCE separation. The best mobile phase for isocratic HPLC was 0.03% TFA-acetonitrile (82:18, v/v). Data obtained with HPCE are in good accordance with those from HPLC; HPCE, however, is clearly more rapid and simple to perform.

Quantitation of clobazam in human plasma using high-performance liquid chromatography.

A rapid and simple reversed-phase high-performance liquid chromatographic (HPLC) method for the determination of clobazam concentrations in human blood samples is developed and validated. Solid-phase column extraction is performed to clean up blood samples before running the analytical HPLC system. The chromatography is isocratic with a mobile phase consisting of acetonitrile (20%, v/v), methanol (23%, v/v), and 0.1 M potassium hydrogen phosphate buffer (pH 3.6; 57%, v/v) at a constant flow rate of 2 mL/min. Clobazam is detected at 226 nm. Chromatography is completed within less than 25 min. The recovery rate is greater than 95% and linear over a wide range of drug concentrations. The intra-assay coefficient of variation percentage varies between 4.3 and 12. This method is used for therapeutic drug monitoring in patients undergoing antiepileptic therapy with clobazam. Plasma levels of clobazam ranged from 21 to 663 ng/mL. Other antiepileptic compounds, such as clonazepam and phenobarbital, did not interfere with the detection of clobazam.

Metabolism of caffeic acid in the isolated perfused rat liver.

Caffeic acid as well as its oxidation products exert a spectrum of biological effects in in vitro testing. To get an idea of the amount as well as the mode of biotransformation, caffeic acid metabolism was investigated by means of the isolated perfused rat liver. The first-pass effect was not pronounced: 93.3% of caffeic acid dose appeared unchanged after one liver passage. Products of caffeic acid oxidation (cyclolignan derivatives) as well as ferulic and isoferulic acid as methylation products were found in the perfusion medium. In addition, a cyclization product, esculetin, was observed. In the bile, mainly glucuronides as well as sulfates of caffeic acid could be determined. Thus, oxidation products and other metabolites formed by liver metabolism can be responsible for the biological effects in vivo.

The effect of Chelidonium majus herb extract on choleresis in the isolated perfused rat liver.

The total ethanolic extract, the phenolic and the alkaloidal fraction of the herb of Chelidonium majus L. (Papaveraceae) were tested for their choleretic activity using the isolated perfused rat liver. The total extract significantly caused chloresis by increasing the bile acid independent flow (BAIF); the observed weak activity of both fractions, tested each and as combination, however, was not significant.

Endocrine effects of Lycopus europaeus L. following oral application.

Lycopus extracts are used in folk medicine for the treatment of hyperthyroid symptoms. Diverse effects on the pituitary thyroidal system as well as on the pituitary gonadal system could be confirmed in experimental studies. But till now endocrine effects of Lycopus extracts in experimental animals were observed after parenteral application only. Therefore in this investigation an ethanolic extract of Lycopus europaeus was applied orally to rats, diverse endocrine parameters were measured between 3 and 24 h later and the effects compared to an i.p. treated group. The plant extract given p.o. caused a long lasting (for a period of more than 24 h) decrease of T3 levels, presumably as a consequence of a reduced peripheral T4 deiodination. A pronounced reduction of T4 and thyroid stimulating hormone (TSH) concentrations was observed 24 h after application of the test solution by gavage. The luteinizing hormone (LH) decrease as well as the TSH decrease, which was pronounced in spite of reduced T4 and T3 levels indicate a central point of attack of the plant extract. Differences in the biological activity in dependence on the route of application may be explained e.g. by differences in absorption of plant constituents.

Mechanisms of the contractile effects of levosimendan in the mammalian heart.

In spontaneously beating guinea pig right atria, levosimendan (LS, or R-[[-(1,4,5,6-tetrahydro-4-methyl-6-oxo-3-pyridazinyl)- phenyl]-hydrazono]propanedinitrile) exerted a positive chronotropic effect starting at 0.1 microM. In electrically driven guinea pig left atria, LS (0.1-10 microM) increased force of contraction without changing time parameters of contraction. In electrically driven right papillary muscles, LS (0.1-10 microM) enhanced force of contraction without affecting time parameters of contraction. The maximal effect on force of contraction at 10 microM amounted to 130 +/- 8.6% of predrug value. The positive inotropic effect of LS in papillary muscles was greatly diminished by additionally applied carbachol. In [32P]-labeled guinea pig ventricular cardiomyocytes, LS increased the phosphorylation state of phospholamban, the inhibitory subunit of troponin and C-protein. The maximal effect at 1 microM amounted to 134 +/- 8.6%, 124 +/- 4.2% and 121 +/- 8% of control for phospholamben, the inhibitory subunit of troponin and C-protein, respectively. LS (1 microM) increased cAMP content from 6.3 +/- 0.3 to 8.1 +/- 0.3 pmol/mg protein in guinea pig ventricular cardiomyocytes. Furthermore, whole-cell patch-clamp studies were performed in guinea pig ventricular cardiomyocytes. In this setup, 10 microM LS increased the amplitude of L-type Ca++ current to 402 +/- 86% of predrug value.

In vivo isoproterenol treatment leads to downregulation of the mRNA encoding the cAMP response element binding protein in the rat heart.

The expression of different myocardial regulatory proteins is altered in human heart failure, e.g., beta 1-adrenoceptors, G-proteins and others. Similar changes in rats after 4 days treatment with isoproterenol led to the hypothesis of the cAMP pathway involved in these changes. In different cell types cAMP-dependent transcriptional activation is mediated by the cAMP-response element binding protein (CREB) which was recently shown to be expressed and phosphorylated in the human heart. Here, by the reverse transcriptase-polymerase chain reaction two alternatively spliced isoforms of CREB mRNA were found to be expressed in rat ventricles. Both isoforms were down-regulated in the ventricles of rats treated in vivo with isoproterenol (2.4 mg/kg per day) for 4 days proposing a possible mechanism involved in expressional changes mentioned above.

Identification and expression of a novel isoform of cAMP response element modulator in the human heart.

In end-stage human heart failure, excessive beta-adrenergic stimulation of the cAMP-dependent signaling pathway due to enhanced endogenous catecholamines is hypothesized to contribute to expressional alterations of myocardial regulatory proteins. The cAMP response element modulator (CREM) regulates the transcription of cAMP-responsive genes and might be involved in the regulation of cardiac gene expression. Using the reverse transcription polymerase chain reaction, we identified a novel CREM mRNA, CREM-Ib deltaC-X, in the human heart. Overexpression of CREM-Ib deltaC-X decreased cAMP response element (CRE) -mediated gene transcription in HIT-T15 cells, and this activity was assigned to the part of the sequence encoding putative internally translated proteins. Two of three possible internally translated proteins were immunologically identified in cells overexpressing CREM-Ib deltaC-X tagged with the hemagglutinin epitope of the influenza virus. Both proteins were expressed in bacteria and showed CRE-specific DNA binding, formation of heterodimers with the cAMP response element binding protein (CREB), and inhibition of CREB's binding to the CRE. CREM expression was detected on the mRNA and protein levels in the human heart. We conclude that CREM-Ib deltaC-X generates internally translated repressors of CRE-mediated gene transcription, suggesting the first example for the existence and function of human cardiac CREM.

Contractility and inhibition of protein phosphatases by cantharidin.

1. Cantharidin is a natural defensive toxicant produced by blister beetles. 2. Cantharidin shares structural similarity with highly toxic commercial herbicides (e.g., endothall, endothall anhydride and endothall thioanhydride). 3. Cantharidin inhibits the activity of purified catalytic subunits of serine/threonine protein phosphatases (PP) type 1 and type 2A. 4. Cantharidin increases force of contraction in isolated myocardial and vascular preparations. 5. Cantharidin enhances the phosphorylation state of myocardial and vascular regulatory proteins. 6. Cantharidin is a valuable tool for studying the function of PP in regulatory phosphorylation-dephosphorylation events.

Postnatal changes in contractile time parameters, calcium regulatory proteins, and phosphatases.

Compared with isolated electrically driven neonatal ventricular preparations, the total time of contraction, the time to peak tension, and the time of relaxation were decreased to approximately 50% in adult ventricular preparations. The expression of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) was increased to 133% at the protein level and to 154% at the mRNA level in adult vs. neonatal ventricular preparations, whereas phospholamban was unchanged at both the protein and mRNA levels. Moreover, Ca2+ uptake was increased to 180% in adult vs. neonatal ventricular preparations. Phospholamban phosphorylation was enhanced in adult vs. neonatal ventricular preparations. In adult ventricular preparations, phosphatase activity was reduced to 53% of neonatal preparations, the protein levels of the immunologically detectable catalytic subunits of protein phosphatase types 1 and 2A were reduced to 28 and 61% of neonatal preparations, respectively, and the mRNA levels of type 1alpha, 1beta, 1gamma, 2Aalpha, and 2Abeta phosphatase isoforms were decreased to 69, 68, 54, 67, and 63%, respectively. We conclude that in the adult rat heart, the shortened time parameters of contraction can be explained by an elevated expression of SERCA. In addition, an increased phosphorylation state of phospholamban due to reduced phosphatase activity may be involved.

cAMP response element binding protein is expressed and phosphorylated in the human heart.

BACKGROUND: In end-stage failing human hearts and in rat hearts after prolonged in vivo beta-adrenergic treatment, several proteins involved in the cAMP-dependent signal transduction are altered on the protein, mRNA, or transcriptional level, eg, beta-adrenoceptors, G-proteins, or proteins of Ca2+ homeostasis. In many tissues, cAMP-dependent transcriptional regulation occurs through the cAMP response element binding protein (CREB) and related transcription factors binding as dimers to cAMP response elements (CREs) in the promoter regions of regulated genes. METHODS AND RESULTS: To investigate a possible role of CREB in the human heart, nuclear protein of explanted failing and nonfailing human hearts was used to test for CRE specific binding properties in gel mobility shift assays. CRE specific binding was found in competition studies, and CREB and its phosphorylated form were immunologically identified in supershift experiments. The alternatively spliced CREB isoforms CREB327 and CREB341 were found to be expressed on the mRNA level by the reverse transcriptase-polymerase chain reaction. CONCLUSIONS: We conclude that in the failing and nonfailing human heart, CREB is expressed on the protein and mRNA levels and that CREB is phosphorylated and able to bind to CREs, indicating a functional role of CREB in the human heart.

Quantification of the cAMP response element binding protein in ventricular nuclear protein from failing and nonfailing human hearts.

Alterations in the expression of myocardial regulatory proteins (e.g. beta-adrenoceptor, inhibitory G-proteins) in human heart failure are associated with excessive stimulation of the cAMP signalling pathway by endogenous catecholamines. The transcription factor cAMP response element binding protein (CREB) mediates cAMP-dependent transcriptional activation and is expressed in the human heart. Here, CREB protein was immunologically quantified in ventricular nuclear protein preparations from nonfailing donor hearts (n = 8) and from failing hearts transplanted due to dilative (n = 10) or ischemic cardiomyopathy (n = 6). CREB expression was unchanged in ventricular nuclei from failing hearts compared to the nonfailing controls suggesting that expressional alterations in human heart failure cannot be explained by altered expression of CREB.