Biomedical subjects

Robert H G Schwinger

Publications and source records attributed to Robert H G Schwinger.

At least 19 recordsLinked to original sources

PubMed2006

Altered tension cost in (TG(mREN-2)27) rats overexpressing the mouse renin gene.

The present study aimed to characterize cardiac hypertrophy induced by activation of the renin-angiotensin system in terms of functional alterations on the level of the contractile proteins, employing transgenic rats harboring the mouse renin gene (TGR(mREN2)27). Ca2+-dependent tension and myosin ATPase activity were measured in skinned fiber preparations obtained from TGR(mREN2)27 and from age-matched Sprague-Dawley rats (SPDR). Western blots for troponin I (TnI) and troponin T (TnT) were performed and the phosphorylation status of TnI were evaluated in myocardial preparations. TnT and myosin heavy chain (MHC) isoforms were analyzed by RT-PCR. The pCa/tension relationship was shifted to the right in TGR(mREN2)27 compared to SPDR as indicated by increased Ca2+-concentrations required for half maximal activation of tension (SPDR 5.80, 95% confidence limits 5.77-5.82 vs. TGR(mREN2)27 5.69, 95% confidence limits 5.67-5.72, pCa units), while maximal developed tension was unaltered. Even more pronounced was the shift in the relationship between pCa and myosin-ATPase (SPDR 6.01, 95% confidence limits 5.99-6.03 vs. TGR(mREN2)27 5.77, 95% confidence limits 5.73-5.79, pCa units). The maximal myosin-ATPase activity was reduced in TGR(mREN2)27 compared to SPDR, respectively (211.0 +/- 28.77 micromol ADP/s vs. 271.6 +/- 43.66 micromol ADP/s, P < 0.05). Tension cost (ATPase activity/tension) was significantly reduced in TGR(mREN2)27. The beta-MHC expression was significantly increased in TGR(mREN2)27. There was no isoform shift for TnT (protein and mRNA), as well as TnI, and no alteration of the phosphorylation of TnI in TGR(mREN2)27 compared to SPRD. The present study demonstrates that cardiac hypertrophy, induced by an activation of the renin-angiotensin system, leads to adapting alterations on the level of the contractile filaments, which reduce tension cost.

Animals↗

PubMed2006

Increased Ca2+ sensitivity and protein expression of SERCA 2a in situations of chronic beta3-adrenoceptor deficiency.

This study investigated the influence of chronic beta(3)-adrenoceptor deficiency on myocardial function. Therefore, we investigated Ca(2+)-regulatory proteins, SERCA 2a activity, and myofibrillar and mitochondrial function in hearts of wild-type (WT, n=7) and beta(3)-adrenoceptor knockout mice (beta(3)-KNO, n=7). Morphometric heart analysis showed no difference between WT and beta(3)-KNO. No alterations were observed for the protein expression of the ryanodine receptor or phospholamban. However, in beta(3)-KNO mice, protein expression of SERCA 2a and phospholamban phosphorylation were significantly increased. These changes were accompanied by an increased SERCA 2a activity in beta(3)-KNO. Alterations in phospholamban phosphorylation were independent of alterations in beta(1)/beta(2)-adrenoceptor distribution and protein expression of G proteins in beta(3)-KNO. Measurement of myofibrillar Ca(2+) sensitivity showed no difference in the Ca(2+)/force relation for WT and beta(3)-KNO. The same seems to hold true for mitochondrial function since the protein expressions of cytochrome c, uncoupling protein 3 and cytochrome c oxidase subunit IV were similar in WT and beta(3)-KNO. The conclusion is that depression of beta(3)-adrenergic stimulation may modulate the protein expression of SERCA 2a and phospholamban phosphorylation, thereby improving sarcoplasmic reticulum Ca(2+) uptake. Thus, beta(3)-adrenergic depression may be a therapeutic aim in situations of impaired SERCA 2a activity, e.g. for the treatment of heart failure.

Animals↗

PubMed2006

Effective engraftment but poor mid-term persistence of mononuclear and mesenchymal bone marrow cells in acute and chronic rat myocardial infarction.

Bone marrow cells are used with promising results for cell therapy after myocardial infarction (MI). We determined the survival and organ distribution of transplanted mononuclear (MNC) or mesenchymal (MSC) bone marrow cells, and the influence of cell type, cell number and application time. MNC and MSC (male Fischer 344 rats) were injected into the border zone of MI (syngeneic females) immediately or 7 days after LAD ligation (10(5) or 10(6) cells, 50 microl). After 0 h, 48 h, 5 days, 3 weeks and 6 weeks, DNA of heart, lung, liver, spleen, kidney, blood, bone marrow, brain and skeletal muscle was isolated and the number of donor cells determined by quantitative real-time PCR with Y-chromosome specific primers (each n>or=4). The percentage of donor-cells in the heart decreased rapidly from 34-80% of injected cells (0 h) to 0.3-3.5% (6 weeks) independent from cell type, number and application time. The absolute number increased after increasing injected cell number (10(6) vs. 10(5)). In the lung, MNC and MSC were found at 0 h (126+/-48 and 140+/-3 per million organ cells), but in liver and kidney, only few. At 48 h and 6 weeks, an increasing number of MNC, but not MSC, were detected in the spleen (6 weeks, 602+/-173 per million organ cells vs. 95+/-50 in the heart, P=0.02). In all other organs, only few or no grafted cells of either cell type were detected at these times. Organ distribution was independent from injection time. The low survival of grafted cells may limit their therapeutic impact, while their distribution to other organs must be considered in all cell therapy applications.

Acute Disease↗

PubMed2006

Alterations of tension-dependent ATP utilization in a transgenic rat model of hypertrophic cardiomyopathy.

Although it is established that familial hypertrophic cardiomyopathy (FHC) is caused by mutations in several sarcomeric proteins, including cardiac troponin T (TnT), its pathogenesis is still not completely understood. Previously, we established a transgenic rat model of FHC expressing a human TnT molecule with a truncation mutation (DEL-TnT). This study investigated whether contractile dysfunction and electrical vulnerability observed in DEL-TnT rats might be due to alterations of intracellular Ca(2+) homeostasis, myofibrillar Ca(2+) sensitivity, and/or myofibrillar ATP utilization. Simultaneous measurements of the force of contraction and intracellular Ca(2+) transients were performed in right ventricular trabeculae of DEL-TnT hearts at 0.25 and 1.0 Hz. Rats expressing wild-type human TnT as well as nontransgenic rats served as controls. In addition, calcium-dependent ATPase activity and tension development were investigated in skinned cardiac muscle fibers. Force of contraction was significantly decreased in DEL-TnT compared with nontransgenic rats and TnT. Time parameters of Ca(2+) transients were unchanged at 0.25 Hz but prolonged at 1.0 Hz in DEL-TnT. The amplitude of the fura-2 transient was similar in all groups investigated, whereas diastolic and systolic fura-2 ratios were found elevated in rats expressing nontruncated human troponin T. In DEL-TnT rats, myofibrillar Ca(2+)-dependent tension development as well as Ca(2+) sensitivity of tension were significantly decreased, whereas tension-dependent ATP consumption ("tension cost") was markedly increased. Thus, a C-terminal truncation of the cardiac TnT molecule impairs the force-generating capacity of the cycling cross-bridges resulting in increased tension-dependent ATP utilization. Taken together, our data support the hypothesis of energy compromise as a contributing factor in the pathogenesis of FHC.

Adenosine Triphosphate↗

PubMed2006

No evidence of myocardial restoration following transplantation of mononuclear bone marrow cells in coronary bypass grafting surgery patients based upon cardiac SPECT and 18F-PET.

BACKGROUND: We tested the hypothesis, that intramyocardial injection of mononuclear bone marrow cells combined with coronary artery bypass grafting (CABG) surgery improves tissue viability or function in infarct regions with non-viable myocardium as assessed by nuclear imaging techniques. METHODS: Thus far, 7 patients (60 +/- 10 [SD] years) undergoing elective CABG surgery after a myocardial infarction were included in this study. Prior to sternotomy, bone marrow was harvested by sternal puncture. Mononuclear bone marrow cells were isolated by gradient centrifugation and resuspended in 2 ml volume of Hank's buffered salt solution. At the end of CABG surgery 10 injections of 0.2 ml each were applied to the core area and borderzones of the infarct. Global and regional perfusion and viability were evaluated by ECG-gated 99mTc-tetrofosmin myocardial single-photon emission computed tomograph (SPECT) imaging and 18F-fluorodeoxyglucose positron emission tomography (FDG-PET) in all study patients < 6 days before and 3 months after the intervention. RESULTS: Non-viable segments indicating transmural defects were identified in 5 patients. Two patients were found to have non-transmural defects before surgery. Concomitant surgical revascularisation and bone marrow cell injection was performed in all patients without major complications. The median total injected mononuclear cell number was 7.0 x 10(7) (range: 0.8-20.4). At 3 months 99mTc-tetrofosmin SPECT and 18F-FDG-PET scanning showed in 5 patients (transmural defect n = 4; non-transmural defect n = 1) no change in myocardial viability and in two patients (transmural defect n = 1, non-transmural defect n = 1) enhanced myocardial viability by 75%. Overall, global and regional LV ejection fraction was not significantly increased after surgery compared with the preoperative value. CONCLUSION: In CABG surgery patients with non-viable segments the concurrent use of intramyocardial cell transfer did not show any clear improvement in tissue viability or function by means of non-invasive bioimaging techniques.

Journal Article↗

PubMed2006

Mesenchymal stem cells transmigrate over the endothelial barrier.

Mesenchymal stem cells (MSCs) seem to be a useful tool for cellular therapy in injured tissues, e.g. myocardial infarction or cardiomyopathies resulting in heart failure. For therapeutic approaches it is crucial that MSCs cross the endothelial barrier especially in intravascular or rather intracoronary application. Until today little is known about MSCs transmigrating across the endothelium. We performed co-culture experiments of MSCs on an endothelial monolayer to analyse direct interactions. An increasing flattened morphology of the MSCs was followed by a total integration into the monolayer after 2h. We repeated these experiments in isolated heart perfusions with gold-labelled MSCs. Using electron microscopy we detected MSCs exhibited direct cell-cell contacts. Tight junctions between the endothelial cells became abolished resulting in a distinct split between the cells. MSCs developed tight cell-cell contacts and became integrated into the endothelial wall of the capillary vessel. Finally, using confocal laser scanning microscopy, we assessed the ability of the MSCs to fully pass the endothelial barrier. Within the first 30 min, 30+/-8% of MSCs transmigrated, increasing to about half at 60 min (50+/-8%), whereas after 120 min the rate remained nearly unchanged (53+/-10%). This work demonstrates the capability of MSCs for transendothelial migration. Moreover we showed that the vast majority of MSCs migrated within 30 min, an important finding for the exposure times in clinical settings.

Animals↗

PubMed2006

ENOS is not activated by nebivolol in human failing myocardium.

Nebivolol is a highly selective beta(1)-adrenoceptor blocker with additional vasodilatory properties, which may be due to an endothelial-dependent beta(3)-adrenergic activation of the endothelial nitric oxide synthase (eNOS). beta(3)-adrenergic eNOS activation has been described in human myocardium and is increased in human heart failure. Therefore, this study investigated whether nebivolol may induce an eNOS activation in cardiac tissue. Immunohistochemical stainings were performed using specific antibodies against eNOS translocation and eNOS serine(1177) phosphorylation in rat isolated cardiomyocytes, human right atrial tissue (coronary bypass-operation), left ventricular non-failing (donor hearts) and failing myocardium after application of the beta-adrenoceptor blockers nebivolol, metoprolol and carvedilol, as well as after application of BRL 37344, a specific beta(3)-adrenoceptor agonist. BRL 37344 (10 microM) significantly increased eNOS activity in all investigated tissues (either via translocation or phosphorylation or both). None of the beta-blockers (each 10 microM), including nebivolol, increased either translocation or phosphorylation in any of the investigated tissues. In human failing myocardium, nebivolol (10 microM) decreased eNOS activity. In conclusion, nebivolol shows a tissue-specific eNOS activation. Nebivolol does not activate the endothelial eNOS in end-stage human heart failure and may thus reduce inhibitory effects of NO on myocardial contractility and on oxidative stress formation. This mode of action may be of advantage when treating heart failure patients.

Adrenergic beta-Antagonists↗

PubMed2006

[Therapy of heart failure in the elderly].

Chronic heart failure is one of the most common diseases in internal medicine. Systolic and diastolic heart failure can be distinguished. According to the guidelines therapy consists of beta-blockers, ACE inhibitors/AT1 receptor antagonists, diuretics, aldosterone antagonists, and glycosides. Older patients should also be treated following the guidelines. However, side effects and interactions between drugs are more frequent and require special attention. The majority of concomitant diseases are no contraindication to heart failure treatment.

Aged↗

PubMed2006

[Mesenchymal stem cells for cardiac regeneration].

Since physiological regeneration of cardiac muscle is limited, scar tissue replaces damaged myocardial tissue leading to impaired cardiac function. Whether stem cell therapy can prevent this process and regenerate cardiac tissue is the issue of many research projects at present. Some studies investigate mesenchymal stem cells (MSC) from bone marrow, which are known to have the potential to differentiate into various tissues. Under certain conditions the formation of cardiomyocytes was observed in vitro and in vivo, which, however, is a controversial issue. Nevertheless many investigators found an improvement of cardiac function after transplantation of MSC into damaged heart tissue. Since only few cells survive mid-term after transplantation, the impact of real cell replacement might be limited. Instead of or besides the replacement of lost cells by transdifferentiated MSC, the secretion of paracrine factors by MSC may be important for functional improvement. Thus, MSC show promising experimental results, and may be of therapeutic value for heart disease in the future. However, there are many unanswered questions and problems, which need to be resolved before.

Animals↗

PubMed2006

[Reverse remodeling of the intracellular Ca(2+)-homeostasis: new concepts of pathophysiology and therapy of heart failure].

Cardiac contraction is dependent on a rapid alteration of the intracellular Ca(2+) concentration, especially the Ca(2+) released during systole. In end-stage heart failure, cardiac contractility is depressed due to alterations in the structure and function of proteins or protein complexes. Over recent years, new insights have been obtained regarding the regulation of the intracellular Ca(2+) homeostasis and its pathophysiological alteration in end-stage heart failure. This review focuses on the mechanisms involved in the release of Ca(2+) from the sarcoplasmic reticulum (SR) during systole via the ryanodine receptors and the Ca(2+)-uptake into the SR by the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA 2a). In addition, new therapeutic options will be introduced which may be of importance for the treatment of heart failure patients.

Calcium↗

PubMed2006

[On the function of beta3-adrenoceptors in the human heart: signal transduction, inotropic effect and therapeutic prospects].

Beta-adrenergic stimulation is an important regulatory mechanism of cardiac function. Next to beta1- and beta2-adrenoceptors, the expression of a third beta-adrenoceptor population, the beta3-adrenoceptor, has recently been evidenced in the human heart. Stimulation of cardiac beta3-adrenoceptors leads to a decrease in contractility via a release of nitric oxide (NO). In this context, different molecular mechanisms of endothelial nitric oxide synthase (eNOS) activation have been uncovered to occur as a consequence of beta3-adrenergic stimulation. In both nonfailing and failing myocardium, beta3-adrenergic stimulation may have a protective effect against excessive chatecolaminergic stimulation as it occurs during somatic and mental stress and during heart failure. For this reason, the beta3-adrenoceptor is discussed as a possible target for the pharmacological therapy of heart failure.

Adrenergic beta-3 Receptor Agonists↗

PubMed2006

Crataegus special extract WS 1442 induces an endothelium-dependent, NO-mediated vasorelaxation via eNOS-phosphorylation at serine 1177.

PURPOSE: This study investigates the influence of WS(R) 1442, a special extract of Crataegus leaves with flowers, on the relaxation of rat aorta and human mammarian artery (coronary bypass patients). METHODS: Experiments were performed in the presence and absence (mechanical disruption) of endothelium. In addition, we investigated three fractions of WS(R) 1442 (fraction A: lipophilic, containing flavonoids and oligomeric procyanidins (OPC), fraction B: hydrophilic, containing flavonoids and low molecular weight OPC, fraction C: hydrophilic, essentially flavonoid-free and rich in high molecular weight OPC). RESULTS: WS 1442 induced a concentration-dependent vasodilation in isolated vessel rings that had been precontracted by 10 microM phenylephrine (concentration for halfmaximal relaxation (IC(50)): rat: 15.1 +/- 0.6 microg/ml (n = 7), human: 19.3 +/- 3.4 microg/ml (n = 6)). The maximal vasorelaxation induced after application of 100 microg of WS 1442 was 75.0 +/- 5.7% (rat) and 79.2 +/- 5.8% (human) of the papaverine (0.1 mM)-induced vasodilation. If the experiments were performed in the presence of L-nitroarginine methylester (10 microM, eNOS-inhibition) or after mechanical disruption of the endothelium, no vasorelaxation was observed in the presence of WS 1442. The vasorelaxant properties of WS 1442 were mediated by fraction C. WS 1442 induced an NO-liberation from human coronary artery endothelial cells as measured by diaminofluorescein. WS 1442 induced eNOS-activation was due to a phosphorylation at serine 1177. No eNOS-translocation or phosphorylation at serine 114 or threonine 495 was observed after application of WS 1442. CONCLUSIONS: It is concluded that WS 1442, induces an endothelium-dependent, NO-mediated vasorelaxation via eNOS phosphorylation at serine 1177.

Adult↗

PubMed2006

Reduced sarcoplasmic reticulum Ca2+ -ATPase activity and dephosphorylated phospholamban contribute to contractile dysfunction in human hibernating myocardium.

Human hibernating myocardium (HHM) is characterized by reversible contractile dysfunction during chronic ischemia. A disturbed calcium-homeostasis is a decisive factor for reduced functional capacity in heart diseases. We therefore investigated calcium-handling proteins in HHM. In 12 patients suffering from multi-vessel coronary artery disease and contractile dysfunction with indication for bypass surgery, HHM was detected preoperatively by thallium scintigraphy, radionuclide ventriculography and dobutamine echocardiography. Transmural biopsies of these regions were taken and analyzed by immunohistochemistry and electron microscopy. Furthermore, SR-calcium ATPase (SERCA2a), phospholamban (PLN), the phosphorylated forms of PLN (PLN-Ser16, PLN-Thr17) as well as sodium-calcium exchanger (NCX) and ryanodine receptor (RyR2) were investigated by RT-PCR and Western-blotting. Additionally, SERCA2a activity was measured by an enzyme-coupled assay. In all patients complete functional recovery could be documented 3 months after revascularization by repeating all preoperative investigations. In HHM maximal SERCA2a activity was significantly reduced (HHM: 424.5 +/- 33.9, control: 609.0 +/- 48.5 nmol ATP mg protein(-1) min(-1), p <or= 0.05), whereas SERCA2a protein levels were unchanged. mRNA levels (HHM: 1.36 +/- 0.08 vs. control: 0.78 +/- 0.04, p <or= 0.05) and protein amount (HHM:1.67 +/- 0.14 vs. control: 1.00 +/- 0.04, p <or= 0.05) of PLN (A1) were increased resulting in an increased PLN:SERCA2a-ratio. PLN-Ser16 (HHM: 0.60 +/- 0.08 vs. control: 1.00 +/- 0.11, p <or= 0.05) and PLN-Thr17 (HHM: 0.63 +/- 0.11 vs. control: 1.00 +/- 0.06, p <or= 0.05) phosphorylation was significantly decreased. RyR2 and NCX showed no significant alteration. In HHM a decreased activity of SERCA2a due to an impaired phosphorylation of PLN contributes to contractile dysfunction. The increase in the relative ratio of PLN/SERCA2a leads to a decreased calcium affinity of SERCA2a.

Aged↗

PubMed2006

Mechanisms underlying nebivolol-induced endothelial nitric oxide synthase activation in human umbilical vein endothelial cells.

1. Nebivolol (NEB) has been shown to be a selective blocker of beta1-adrenoceptors with additional vasodilating properties that are mediated, at least in part, by an endothelial-dependent liberation of nitric oxide (NO). In the present study, we investigated the underlying mechanisms of NEB-induced vasodilation. 2. Immunohistochemical staining of endothelial nitric oxide synthase (eNOS) was performed in the absence and presence of NEB in human umbilical vein endothelial cells (HUVEC). In addition, we measured the release of nitric oxide (NO) using diaminofluorescein. Metoprolol (MET) was used for comparison. 3. Nebivolol, but not MET (each at 10 micromol/L), caused a time-dependent increase in NO release from HUVEC, as demonstrated by an increase in DAF fluorescence at 0 versus 10 min (+234 +/- 7 and 55 +/- 22% basal, respectively). Blockade of beta3-adrenoceptors by SR 59230A (1 micromol/L) partially reduced the NEB-induced increase in DAF fluorescence. Complete inhibition of NEB-induced NO liberation was achieved by the simultaneous blockade of beta3-adrenoceptors and oestrogen receptors (with 1 micromol/L ICI 182,780). 4. Application of NEB significantly increased eNOS translocation and serine 1177 phosphorylation of eNOS. However, NEB did not alter eNOS-phosphorylation at threonine 495 and at serine 114. 5. In conclusion, the endothelium-dependent NO liberation induced by NEB is due to stimulation of beta3-adrenoceptors and oestrogen receptors and coincides with eNOS translocation and a phosphorylation at eNOS-serine 1177. These characteristics of NEB may be beneficial not only when treating patients suffering from cardiovascular disease, but may also prevent further deterioration of endothelial dysfunction.

Adrenergic beta-Antagonists↗

PubMed2006

Effect of prophylactic bisoprolol plus magnesium on the incidence of atrial fibrillation after coronary bypass surgery: results of a randomized controlled trial.

OBJECTIVE: Bisoprolol, a highly cardioselective beta(1)-blocker, is widely used to treat elderly patients with hypertension, coronary artery disease and heart failure. The current literature lacks evidence regarding its potency to prevent atrial fibrillation (AF) following cardiac surgery. Therefore the aim of this study was to evaluate the efficacy of bisoprolol plus magnesium (Mg) in the prophylaxis of AF after coronary artery bypass graft (CABG) surgery. RESEARCH DESIGN AND METHODS: A total of 100 consecutive patients subjected to elective on-pump CABG (84 men, age 65 +/- 8 [SD] years), with no prior AF history, were randomly assigned to the prophylaxis group (n = 50) receiving after surgery bisoprolol (5 mg/day) plus Mg (intravenous infusion of 2 g of Mg on arrival in the intensive care unit, followed by oral Mg at 1800 mg/day for 1 week), or to the control group (n = 50), receiving no combined study medication but remaining on their preoperative drugs, including beta-blockers. All patients were continuously monitored to identify the onset of AF. RESULTS: In the prophylaxis group the incidence of postoperative AF was significantly lower, with 20% (10 / 50) compared to 42% (21 / 50) among controls (p = 0.030, 95% confidence interval [CI] for absolute risk reduction [ARR], 2-42%). Particularly in the elderly, bisoprolol plus Mg was effective in preventing AF; in the prophylaxis group only six of 36 (17%) patients > or = 65 years of age developed AF, compared to 13 of 20 (65%) in the control group (p < 0.001, 95% CI for ARR, 17-65%). This was associated with significantly (p = 0.022) shorter hospital stays in the prophylaxis group (median of 7 vs. 9 days, 95% CI for difference in medians, 0-3 days). CONCLUSIONS: The combination of bisoprolol plus Mg effectively reduces the incidence of postoperative AF following on-pump CABG, particularly in elderly patients, and is associated with a shorter hospital length of stay.

Aged↗

PubMed2006

Basic fibroblast growth factor controls migration in human mesenchymal stem cells.

Little is known about the migration of mesenchymal stem cells (MSCs). Some therapeutic approaches had demonstrated that MSCs were able to regenerate injured tissues when applied from different sites of application. This implies that MSCs are not only able to migrate but also that the direction of migration is controlled. Factors that are involved in the control of the migration of MSCs are widely unknown. The migratory ability of isolated MSCs was tested in different conditions. The migratory capability was examined using Boyden chamber assay in the presence or absence of basic fibroblast growth factor (bFGF), erythropoietin, interleukin-6, stromal cell-derived factor-beta, and vascular endothelial growth factor. bFGF in particular was able to increase the migratory activity of MSCs through activation of the Akt/protein kinase B (PKB) pathway. The results were supported by analyzing the orientation of the cytoskeleton. In the presence of a bFGF gradient, the actin filaments developed a parallelized pattern that was strongly related to the gradient. Surprisingly, the influence of bFGF was not only an attraction but also routing of MSCs. The bFGF gradient experiment showed that low concentrations of bFGF lead to an attraction of the cells, whereas higher concentrations resulted in repulsion. This ambivalent effect of bFGF provides the possibility to a purposeful routing of MSCs.

Actins↗

PubMed2005

eNOS translocation but not eNOS phosphorylation is dependent on intracellular Ca2+ in human atrial myocardium.

In endothelial cells, two ways of endothelial nitric oxide (NO) synthase (eNOS) activation are known: 1) translocation and 2) Akt-dependent phosphorylation of the enzyme at Ser(1177) (Ser(1177) eNOS). We have recently shown that agonist-induced Ser(1177) eNOS phosphorylation also occurs in human myocardium (10). In this study, we investigated the Ca(2+) dependency of these two mechanisms in human atrium. Therefore, atrial tissue was obtained from patients who underwent coronary artery bypass operations. In immunohistochemical experiments, the translocated form of eNOS and phosphorylated Ser(1177) eNOS were labeled using specific antibodies. eNOS translocation was measured in the absence and presence of the Ca(2+) chelator BAPTA before and after application of BRL 37344 (BRL), a beta(3)-adrenoceptor agonist that increases eNOS activity (34). In the absence of BAPTA, BRL time dependently increased the staining intensity of translocated eNOS, whereas in the presence of BAPTA, this effect was blunted. In contrast, BRL clearly increased the staining of phosphorylated Ser(1177) eNOS even in the presence of BAPTA. This observation was confirmed using Western blot analysis. Using the NO-sensitive dye diaminofluorescein, we have demonstrated that BRL induced a strong NO release. This effect was completely abolished in the presence of BAPTA but was unaffected by LY-292004, an inhibitor of phosphatidylinositol 3-kinase activity and eNOS phosphorylation. Although Ca(2+) dependent, neither the translocation of eNOS nor NO release was changed by the adenylate cyclase activator forskolin. In conclusion, 1) in human atrial myocardium, BRL-induced eNOS translocation but not Ser(1177) eNOS phosphorylation is dependent on intracellular Ca(2+). 2) In atrial myocardium, eNOS-translocation and not Ser(1177) eNOS phosphorylation is responsible for generating the main amount of NO. 3) Although Ca(2+) dependent, eNOS translocation and NO release could not be mimicked by adenylate cyclase activation as a mediator of beta-adrenergic stimulation.

Calcium↗

PubMed2005

Phosphodiesterase type 5 inhibitor sildenafil citrate does not potentiate the vasodilative properties of nebivolol in rat aorta.

BACKGROUND: Sildenafil citrate (SIL) is contraindicated in patients with coronary heart disease who are treated with nitric oxide (NO) donators such as organic nitrates, as it potentiates NO-mediated vasodilation. The present study investigated whether SIL also affects the vasodilatory effects of nebivolol (NEB), a selective beta1-adrenoceptor blocker with an additional, endothelium-dependent NO-liberating property, in comparison to the combination SIL/glycerol trinitrate (GTN). METHODS AND RESULTS: Experiments were performed in isolated vessel rings of rat aorta (Wistar rats, 8-12 weeks), which had been pre-contracted with phenylephrine (10(-5) M). Isometric tension was measured by a force transducer, and cumulative concentration-response curves were obtained for each drug. The rank order of vasodilatory potency as measured by the concentration needed to achieve 50% relaxation (EC50) was GTN (0.08 microM) > SIL (1.25 microM) > or = NEB (3.5 microM). In the presence of both therapeutic (1 nM) and high (1 microM) concentrations of SIL, vasodilation of GTN was potentiated as indicated by a significant increase in vasodilatory potency (EC50 GTN + low SIL: 0.019 microM, EC50 GTN + high SIL: 0.002 microM; both P < 0.01 vs. GTN). In contrast, SIL did not potentiate the vasodilatory effect of NEB (EC50 NEB + low SIL: 5.01 microM, EC50 NEB + high SIL: 3.2 microM; n.s. vs. NEB). CONCLUSIONS: These data demonstrate that SIL does not potentiate NEB-induced vasodilation in vitro. These findings indicate that the interaction between SIL and NO-donators/organic nitrates does not apply to the NO-liberating properties of NEB. Our findings suggest that SIL may safely be used in hypertensive patients treated with NEB.

3',5'-Cyclic-GMP Phosphodiesterases↗