Biomedical subjects

W Schmitz

Publications and source records attributed to W Schmitz.

At least 55 records · Page 3Linked to original sources

PubMed1999

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.

Cardiomyopathies↗

PubMed1999

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.

Chromatography, Affinity↗

PubMed1999

Activity of cAMP-dependent protein kinase and Ca2+/calmodulin-dependent protein kinase in failing and nonfailing human hearts.

OBJECTIVES: A hallmark of human heart failure is prolonged myocardial relaxation. Although the intrinsic mechanism of phospholamban coupling to the Ca(2+)-ATPase is unaltered in normal and failed human hearts, it remains possible that regulation of phospholamban phosphorylation by cAMP-dependent mechanisms or other second messenger pathways could be perturbed, which may account partially for the observed dysfunctions of the sarcoplasmic reticulum (SR) associated with this disease. METHODS: cAMP-dependent protein kinase (PKA) and Ca2+/calmodulin-dependent protein kinase II (CaM kinase) were characterized initially by DEAE-Sepharose chromatography in hearts from patients with end-stage dilated cardiomyopathy. We measured the activity of PKA and CaM kinase in left ventricular tissue of failing (idiopathic dilated cardiomyopathy; ischemic heart disease) and nonfailing human hearts. RESULTS: Basal PKA activity was not changed between failing and nonfailing hearts. One major peak of CaM kinase activity was detected by DEAE-Sepharose chromatography. CaM kinase activity was increased almost 3-fold in idiopathic dilated cardiomyopathy. In addition, hemodynamical data (left ventricular ejection fraction, cardiac index) from patients suffering from IDC positively correlate with CaM kinase activity. CONCLUSIONS: Increased CaM kinase activity in hearts from patients with dilated cardiomyopathy could play a role in the abnormal Ca2+ handling of the SR and heart muscle cell.

Adult↗

PubMed1999

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.

Atrial Natriuretic Factor↗

PubMed1999

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.

Adenosine↗

PubMed1999

Expression of endothelial cell-derived nitric oxide synthase (eNOS) is increased during gastric adaptation to chronic aspirin intake in humans.

BACKGROUND: Gastric adaptation to aspirin is well-documented. However, the mechanisms underlying the reduction of aspirin-induced mucosal damage despite continued ingestion of the drug remain poorly understood. METHODS: Eight healthy volunteers who received aspirin 1 g b.d. for 14 days were compared with eight placebo-dosed controls. Gastroscopy with mucosal biopsy was performed, and gastric mucosal blood flow was measured before and following 3, 7 and 14 days of aspirin treatment. At the same time points, tissue concentration and the content of prostaglandin E2 in the gastric juice were determined and expression of endothelial cell-derived nitric oxide synthase (eNOS) in mucosal biopsies was measured using Western blot analysis. RESULTS: Aspirin-induced mucosal damage that reached a maximum on day 3, declining significantly by day 14. Concomitantly, mucosal blood flow significantly increased on day 3 and returned to initial values on day 14. Aspirin intake led to a significant decrease in prostaglandin E2 concentration in the gastric mucosa and in gastric juice during the whole period of aspirin consumption. eNOS expression started to increase on day 7 in oxyntic mucosa and on day 3 in antral mucosa, reaching its highest values at the end of the consumption of aspirin. CONCLUSIONS: The human gastric mucosa adapts to prolonged aspirin intake, and this is accompanied by an increase in mucosal blood flow and reduced prostaglandin synthesis. Increase of mucosal eNOS expression might compensate for reduced prostaglandin synthesis and be responsible for gastric adaptation to chronic aspirin intake in humans.

Adult↗

PubMed1999

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.

Anti-Anxiety Agents↗

PubMed1999

Identification and characterization of diadenosine 5',5"'-P1,P2 -diphosphate and diadenosine 5',5"'-P1,P3-triphosphate in human myocardial tissue.

We examined whether human cardiac tissue contains diadenosine polyphosphates and investigated their physiological role. Extracts from human cardiac tissue from transplant recipients were fractionated by size exclusion-, affinity-, anion exchange- and reversed-phase chromatography. MALDI-MS analysis of two absorbing fractions revealed molecular masses of 676.2 Da and 756.0 Da. The UV spectra of both fractions were identical to that of adenosine. Postsource decay MALDI mass spectrometry indicated that the molecules with a mass of 676.2 Da and 757.0 Da contained AMP and ATP, respectively. As shown by enzymatic cleavage, both molecules consist of two adenosines interconnected by either two or three phosphates in 5'-positions of the riboses. Two substances can be identified as 5',5"'-P1,P2-diphosphate (Ap2A) and 5',5"'-P1, P3-triphosphate (Ap3A). Ap2A and Ap3A, together with ATP and ADP, are stored in myocardial-specific granules in biologically active concentrations. In the isolated perfused rat heart, Ap2A and Ap3A caused dose-dependent coronary vasodilations. In myocardial preparations, Ap2A and Ap3A attenuated the effect of isoproterenol, exerting a negative inotropic effect. The calcium current of guinea pig ventricular myocytes, stimulated by isoproterenol, was also attenuated by Ap2A and Ap3A. The presence of Ap2A and Ap3A in cardiac-specific granules and the actions of these substances on the myocardium and coronary vessels indicate a role for these substances as endogenous modulators of myocardial functions and coronary perfusion.

Animals↗

PubMed1999

Inotropic effects of diadenosine tetraphosphate in isolated canine cardiac preparations.

We studied the effects of diadenosine tetraphosphate (AP4A) on the force of contraction in canine preparations. The force of contraction was measured in isolated electrically driven (1 Hz) atrial and ventricular cardiac trabeculae from adult dogs. AP4A (100 microM) alone and after prestimulation with 10 nM isoproterenol reduced force of contraction in atrial preparations by approximately 24%. Moreover, AP4A (100 microM) alone and after prestimulation with 10 nM isoproterenol reduced the force of contraction in ventricular preparations by 29 and 29%, respectively. The negative inotropic effects of AP4A were abolished by the A1-adenosine receptor antagonist 1,3-dipropyl-cyclopentyl-xanthine (DPCPX). In summary, in canine myocardium, AP4A alone and after prestimulation with a beta-adrenoceptor agonist exerts negative inotropic effects, which are probably mediated via A1-adenosine receptors.

Animals↗

PubMed1999

Dissociation of the effects of forskolin and dibutyryl cAMP on force of contraction and phospholamban phosphorylation in human heart failure.

Forskolin and dibutyryl cyclic adenosine monophosphate (cAMP) stimulate force of contraction independent of beta-adrenoceptor stimulation. We studied their effects on force of contraction and phosphorylation of regulatory proteins in isolated electrically driven trabeculae carneae from failing human ventricles. The phosphorylation state of the regulatory protein phospholamban was studied because its phosphorylation usually faithfully follows contractility. For comparison, the phosphorylation state of the inhibitory subunit of troponin was studied. The phosphorylation state was inferred from in vitro phosphorylation of homogenates with cAMP-dependent protein kinase in the presence of radioactive gamma[32P]ATP Proteins were separated by electrophoresis, and radioactivity in the proteins of interest was quantified. The maximal positive inotropic effects occurred at 30 microM forskolin and were attenuated in comparison with the maximal effects to dibutyryl cAMP (1 mM). Both forskolin and dibutyryl cAMP enhanced phospholamban phosphorylation. However, phospholamban phosphorylation in intact trabeculae treated with 30 microM forskolin and 1 mM dibutyryl cAMP was comparable. It is suggested that phospholamban phosphorylation can be dissociated from inotropy at least in isolated trabeculae from failing human hearts.

Adolescent↗

PubMed1999

The protein phosphatase inhibitor cantharidin alters vascular endothelial cell permeability.

In this study, we characterized the effects of the protein phosphatases type 1 (PP 1) and type 2A (PP 2A) inhibitor cantharidin in endothelial cells. We identified catalytic subunits of PP 1alpha, PP 2Aalpha, and PP 2Abeta immunologically in bovine aortic endothelial cells. Moreover, we detected mRNAs coding for catalytic subunits of PP 1alpha, PP 1beta, and PP 2Aalpha by hybridization with specific DNA probes in total RNA from these cells. Okadaic acid and cantharidin inhibited the activities of catalytic subunits of PP 1 (okadaic acid, 0.01-1 microM; cantharidin, 1-100 microM) and PP 2A (okadaic acid, 0.1 nM to 1 microM; cantharidin, 0.1-100 microM) separated by column chromatography in a concentration-dependent manner. Moreover, cantharidin (1 microM to 1 mM) increased the phosphorylation state of endothelial proteins including the regulatory light chains of myosin without affecting cytosolic calcium concentrations. Cantharidin (5-100 microM) increased the permeability of cultured endothelial cells in a time- and concentration-dependent manner. We suggest that inhibition of PP 1 and PP 2A activities by cantharidin increases endothelial permeability by enhancing the phosphorylation state of endothelial regulatory proteins. Thus, cantharidin might be a useful tool to study the function of protein phosphatases in endothelial barrier function.

Animals↗

PubMed1999

Inotropic effects of diadenosine tetraphosphate (AP4A) in human and animal cardiac preparations.

Diadenosine tetraphosphate (AP4A) is an endogenous compound and exerts diverse physiological effects in animal systems. However, the effects of AP4A on inotropy in ventricular cardiac preparations have not yet been studied. The effects of AP4A on force of contraction (FOC) were studied in isolated electrically driven guinea pig and human cardiac preparations. Furthermore, the effects of AP4A on L-type calcium current and [Ca]i were studied in isolated guinea pig ventricular myocytes. In guinea pig left atria, AP4A (0.1-100 microM) reduced FOC maximally by 36.5 +/- 4.3%. In guinea pig papillary muscles, AP4A (100 microM) alone was ineffective, but reduced isoproterenol-stimulated FOC maximally by 29.3 +/- 3.4%. The negative inotropic effects of AP4A in atria and papillary muscles were abolished by the A1-adenosine receptor antagonist 1, 3-dipropyl-cyclopentylxanthine. In guinea pig ventricular myocytes, AP4A (100 microM) attenuated isoproterenol-stimulated L-type calcium current and [Ca]i. In human atrial and ventricular preparations, AP4A (100 microM) alone increased FOC to 158.3 +/- 12.4% and 167.5 +/- 25.1%, respectively. These positive inotropic effects were abolished by the P2-purinoceptor antagonist suramin. On the other hand, AP4A (100 microM) reduced FOC by 27.2 +/- 7.4% in isoproterenol-stimulated human ventricular trabeculae. The latter effect was abolished by 1,3-dipropyl-cyclopentylxanthine. In summary, after beta adrenergic stimulation AP4A exerts negative inotropic effects in animal and human ventricular preparations via stimulation of A1-adenosine receptors. In contrast, AP4A alone can exert positive inotropic effects via P2-purinoceptors in human ventricular myocardium. Thus, P2-purinoceptor stimulation might be a new positive inotropic principle in the human myocardium.

Animals↗

PubMed1998

[Vascular ergotism through inhalation of grain dust].

HISTORY AND CLINICAL FINDINGS: For 6 months a 42-year-old farmer without cardiovascular risk factors had been suffering from increasing pain in both feet and calves. Angiography two months apart had demonstrated progressive narrowing of all lower-leg arteries. Pain-free walking had become restricted to 50 m, there were no palpable pulses in the right foot and those in the left foot were markedly reduced. INVESTIGATIONS: Occlusion pressure of the right foot was 55 mm Hg. Repeat angiography showed proximal occlusion of all three lower-leg arteries. Biopsy and biochemical tests excluded degenerative and inflammatory causes of the vascular disease. DIAGNOSIS, TREATMENT AND COURSE: Enquiry of the patient discovered that he had been exposed to ergotamine-containing milling dust in the preparation of rye flour. Inhalational intake of ergotamine was proven by a high plasma ergotamine level. Attempted treatment with prostaglandin E1 (intraarterially for 16 days), doxazosin (2 mg/d) and acetylsalicylic acid 300 mg/d) had only little effect on symptoms. But complete avoidance of exposure to flour dust slowly decreased the plasma level of ergotamine within 4 months, after which all lower-leg arteries had almost completely re-opened. CONCLUSIONS: This is the first reported case proving that chronic ergotamine inhalation can cause ergotism affecting peripheral arteries. As the plasma ergotamine level fell only slowly it must be assumed that ergotamine had accumulated in a, so far unknown, body depot with slow release into the blood.

Administration, Inhalation↗

PubMed1998

Molecular aspects of adrenergic signal transduction in cardiac failure.

Abnormal beta-adrenergic signal transduction and intracellular Ca2+ handling appear to be a major cause of systolic and diastolic dysfunction in humans with heart failure. The precise mechanisms which cause an alteration in Ca2+ handling have been a subject of investigation in recent years. Several lines of evidence suggest that activation of neurohormonal systems plays a central role. Altered Ca2+-handling (increased diastolic concentrations, reduced systolic Ca2+ release) have a strong impact on diastolic and systolic performance of failing hearts. Sarcoplasmic reticulum Ca2+ ATPase is reduced in activity and in steady-state mRNA concentration. The Na+-Ca2+ exchanger is upregulated at the mRNA and protein levels. Phospholamban depends strongly on cAMP-dependent phosphorylation. A strong sympathetic activation has been shown to desensitize the cAMP system. At the receptor level, there is downregulation of beta1-adrenergic receptors. An uncoupling of beta2-adrenoceptors has been attributed to an increased activity and gene expression of beta-adrenergic receptor kinase in failing myocardium, leading to phosphorylation and uncoupling of receptors. Finally, recent evidence suggests that cAMP-dependent transcription mechanisms may play a role during beta-adrenergic stimulation and cardiomyopathy with heart failure - by means of altered actions of cAMP response element binding protein, the cAMP response element modulator, or the activating transcription factor 1. The exact characterization of signal transduction defects could offer novel approaches to the pharmacological treatment of heart failure.

Animals↗

PubMed1998

Expression of calcium regulatory proteins in short-term hibernation and stunning in the in situ porcine heart.

BACKGROUND: Myocardial hibernation and stunning are characterised by a reversible contractile dysfunction during and after ischaemia, respectively. Calcium homeostasis might be disturbed in hibernation and stunning due to altered expression of cardiac proteins involved in calcium handling. METHODS: In enflurane-anaesthetised swine the coronary blood flow through the left anterior descending coronary artery was decreased to reduce regional contractile function (microsonometry) by approximately 50%. In transmural biopsies obtained during ischaemia and reperfusion creatine phosphate as well as the expression of sarcoplasmic reticulum calcium ATPase (SERCA), phospholamban (PLB), calsequestrin (CSQ), and troponin inhibitor (TnI) were determined. RESULTS: During ischaemia creatine phosphate, after an initial reduction, recovered back to control values, and necrosis was absent (hibernation). After 90 min of ischaemia the myocardium was reperfused for 120 min but regional contractile function continued to be depressed (stunning). PLB, SERCA, CSQ, and TnI proteins were unchanged during ischaemia as well as reperfusion. Likewise, levels of PLB and SERCA mRNAs were unchanged. CONCLUSION: It is concluded that other mechanisms than altered expression of these regulating proteins underlie the contractile dysfunction observed during acute ischaemia, short-term hibernation and stunning.

Animals↗

PubMed1998

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.

Animals↗

PubMed1998

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.

Animals↗

PubMed1998

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.

Animals↗