DNA damage-induced histone H1 ubiquitylation is mediated by HUWE1 and stimulates the RNF8-RNF168 pathway.

The DNA damage response (DDR), comprising distinct repair and signalling pathways, safeguards genomic integrity. Protein ubiquitylation is an important regulatory mechanism of the DDR. To study its role in the UV-induced DDR, we characterized changes in protein ubiquitylation following DNA damage using quantitative di-Gly proteomics. Interestingly, we identified multiple sites of histone H1 that are ubiquitylated upon UV-damage. We show that UV-dependent histone H1 ubiquitylation at multiple lysines is mediated by the E3-ligase HUWE1. Recently, it was shown that poly-ubiquitylated histone H1 is an important signalling intermediate in the double strand break response. This poly-ubiquitylation is dependent on RNF8 and Ubc13 which extend pre-existing ubiquitin modifications to K63-linked chains. Here we demonstrate that HUWE1 depleted cells showed reduced recruitment of RNF168 and 53BP1 to sites of DNA damage, two factors downstream of RNF8 mediated histone H1 poly-ubiquitylation, while recruitment of MDC1, which act upstream of histone H1 ubiquitylation, was not affected. Our data show that histone H1 is a prominent target for ubiquitylation after UV-induced DNA damage. Our data are in line with a model in which HUWE1 primes histone H1 with ubiquitin to allow ubiquitin chain elongation by RNF8, thereby stimulating the RNF8-RNF168 mediated DDR.

Adenovirus-based phospholamban antisense expression as a novel approach to improve cardiac contractile dysfunction: comparison of a constitutive viral versus an endothelin-1-responsive cardiac promoter.

BACKGROUND: A decrease in sarcoplasmic reticulum Ca(2+) pump (SERCA2) activity is believed to play a role in the impairment of diastolic function of the failing heart. Because the expression ratio of phospholamban (PL) to SERCA2 may be a target to improve contractile dysfunction, a PL antisense RNA strategy was developed under the control of either a constitutive cytomegalovirus (CMV) or an inducible atrial natriuretic factor (ANF) promoter. The latter is upregulated in hypertrophied and failing heart, allowing "induction-by-disease" gene therapy. METHODS AND RESULTS: Part of the PL cDNA was cloned in antisense and sense directions into adenovectors under the control of either a CMV (Ad5CMVPLas and Ad5CMVPLs, respectively) or ANF (Ad5ANFPLas and Ad5ANFPLs, respectively) promoter. Infection of cultured rat neonatal cardiomyocytes with Ad5CMVPLas reduced PL mRNA to 30+/-7% of baseline and PL protein to 24+/-3% within 48 and 72 hours, respectively. The effects were vector dose dependent. Ad5CMVPLas increased the Ca(2+) sensitivity of SERCA2 and reduced the time to 50% recovery of the Ca(2+) transient. A decrease of PL protein was also achieved by infection with Ad5ANFPLas, and the presence of the hypertrophic stimulus, endothelin-1, led to enhanced downregulation of PL. The adenovectors expressing PL sense RNA had no effect on any of the tested parameters. CONCLUSIONS: Vector-mediated PL antisense RNA expression may become a feasible approach to modulate myocyte Ca(2+) homeostasis in the failing heart. The inducible ANF promoter for the first time offers the perspective for induction-by-disease gene therapy, ie, selective expression of therapeutic genes in hypertrophied and failing cardiomyocytes.

Increased activity of the sarcoplasmic reticular calcium pump in porcine stunned myocardium.

OBJECTIVE: The aim was to determine whether changes in sarcoplasmic reticular Ca2+ transport activity and the degree of phosphorylation of phospholamban of "stunned" myocardium are involved in the reversible depression of contractile function. METHODS: In anaesthetised open chest swine, stunning was induced by subjecting the myocardium perfused by the left anterior descending coronary artery to two cycles of 10 min of occlusion and 30 min of reperfusion. Before and after stunning, systemic haemodynamic variables and regional myocardial function and perfusion were determined, while biopsies were taken for determination of the content of high energy phosphate compounds. Sarcoplasmic reticular function (ATP dependent Ca2+ transport and phosphorylation of phospholamban) of the stunned and control myocardium was determined at the end of the stunning protocol. RESULTS: In the stunned myocardium the segment length shortening decreased from 17.4(SD 4.0)% to 3.5(4.4)%, while perfusion was 38% less than at baseline. ATP and total adenine nucleotide levels of the stunned myocardium were about 35% lower than in the control myocardium, but the energy charge was normal as creatine phosphate levels had increased by 66% over the content determined at baseline. Ca2+ uptake by the sarcoplasmic reticulum isolated from the stunned region was 17% (p < 0.05) higher than Ca2+ uptake from the control region [1240(303) and 1450(280) nmol.min-1.mg-1 protein, respectively]. In the presence of exogenous cyclic AMP dependent protein kinase the amount of 32P incorporated into phospholamban was similar for both myocardial regions. CONCLUSIONS: In this model of stunned porcine myocardium, the phosphorylation state of phospholamban was unchanged, but Ca2+ uptake by the sarcoplasmic reticulum was slightly increased. The results indicate that a change in active Ca2+ transport by the sarcoplasmic reticulum is most likely not to be the principal cause of contractile dysfunction of stunned myocardium.

Uptake of thyroid hormones in neonatal rat cardiac myocytes.

The uptake and metabolism of T3 and T4 were investigated in cardiomyocytes isolated from 2-day-old rats. Myocytes (2-5 x 10(5) cells/well) were cultured for 1 day in medium with 5% horse serum-5% FCS and subsequently for 4 days without serum; in some cases myocytes were cultured with serum throughout the culture period. Experiments were performed at 37 C in medium with 0.5% BSA for measurement of [125I]T3 (200,000 cpm; 200 pM) uptake and with 0.1% BSA for measurement of [125I]T4 (200,000 cpm; 350 pM) uptake. Uptake of [125I]T3, expressed as femtomoles per picomolar concentration of free hormone, with any incubation time between 15 min and 24 h was at least 2-fold higher than that of [125I]T4. Neither T3 nor T4 was deiodinated within 24 h. This was observed in cells cultured in the absence or presence of serum. After 15 min of incubation, [125I]T3 uptake was 0.048 +/- 0.002 fmol/pM free T3 (n = 9), and [125I]T4 uptake was 0.018 +/- 0.003 fmol/pM free T4 (n = 9). Although [125I]T3 uptake was reduced by 31-40% (P < 0.05) by coincubation with 100 nM to 10 microM unlabeled T3, that of [125I]T4 was not affected by 1 nM to 10 microM unlabeled T4, nor was [125I]T3 uptake reduced by 10 microM unlabeled T4. Preincubation (30 min) and incubation (15 min) with 10 microM oligomycin reduced cellular ATP by 56% (P < 0.05) and [125I]T3 uptake by 73% (P < 0.05), but had no effect on [125I]T4 uptake. Similarly, [125I]T3 uptake, but not [125I]T4 uptake, was dependent on temperature and partly dependent on the Na+ gradient, as shown by the inhibitory effect of 10 microM monensin (27%; P < 0.05). The effect of aromatic amino acids (2 mM) on [125I]T3 uptake increased in the order phenylalanine < tyrosine < tryptophan. It is concluded that T3 is taken up in neonatal cardiomyocytes by an energy-dependent carrier-mediated mechanism that is also partly dependent on the Na+ gradient. Such a transport mechanism for T4 is not present in the neonatal heart, but it may appear later during development.

The influence of the chain length of aldehydes on the fluorescence of chromolipids.

Incubation of phosphatidylethanolamine containing liposomes with malondialdehyde and other aldehydes with different chain lengths results in the fluorescence of chromolipids. Relative to malondialdehyde, the fluorescence was greatly enhanced with increasing chain length upon incubation of 2-alkenals with phospholipids. Similar results were found using the total lipid extracted from erythrocyte ghosts. It seems that the hydrophobic character of the aldehydes is important for the amount of fluorescence detected in lipid bilayers.

Effects of nisoldipine on recovery of coronary blood flow, sarcoplasmic reticulum function and other biochemical parameters in post-ischaemic porcine myocardium.

The effects of nisoldipine (0.1 micrograms/kg/min; n = 9) or its solvent (n = 9) were studied in pigs, in which left anterior descending coronary artery (LADCA) blood flow in both groups was reduced to 20% of baseline for 60 min and reperfused for 2 hr. Infusions were started at 30 min of ischaemia and lasted throughout reperfusion. In both groups, flow reduction abolished regional contractile function and caused similar decreases in the level of creatine phosphate (CP; by 70%) and the energy charge (from 0.91 to 0.69), mean arterial blood pressure (by 25%), LVdP/dtmax (by 30%) and cardiac output (by 30%). During ischaemia LADCA blood flow slightly increased (from 14 +/- 8 to 24 +/- 6 mL/min/100 g; P less than 0.05) in the nisoldipine-treated animals, resulting in an increase in CP to 91 +/- 24% of baseline and preventing further decreases in energy charge, as observed in the solvent-treated animals. After 2 hr of reperfusion in neither group return of contractile function of the post-ischaemic myocardium was observed. Post-ischaemic blood flow in the nisoldipine-treated pigs increased from 24 +/- 6 mL/min/100 g to 76 +/- 14 mL/min/100 g and from 19 +/- 6 mL/min/100 g to 41 +/- 6 ml/min/100 g in the solvent-treated animals (P less than 0.05) after 2 hr of reperfusion. Myocardial work was significantly higher in the nisoldipine-treated animals (111 +/- 15 mmHg.L/min as compared to 69 +/- 14 mmHg.L/min in the solvent-treated pigs after 2 hr of ischaemia). The energy charge of the post-ischaemic myocardium was similar for both groups (0.84 +/- 0.02 for the nisoldipine-treated and 0.83 +/- 0.03 for the solvent-treated animals). The rate of sarcoplasmic reticular Ca2+ uptake of the non-ischaemic segment of the nisoldipine-treated animals was 61% higher (P less than 0.05) than that of the solvent-treated animals. In the post-ischaemic myocardium similar rates of Ca2+ uptake were found in both groups, but the activities were markedly lower as compared to the non-ischaemic myocardium. It is concluded that nisoldipine increases blood flow during reperfusion, which may have been caused by coronary vasodilatation. However, attenuation of the "no-reflow" phenomenon also contributed, since more rapid rephosphorylation of ADP leading to an increase in CP during ischaemia may have preserved jeopardized cells. Moreover, nisoldipine increases the sarcoplasmic reticular Ca2+ pump activity independent of ischaemia, which may have contributed in reducing the Ca2+ overload.

The effect of a thiadiazinone derived Ca2+ sensitizer on the responsiveness of Mg(2+)-ATPase to Ca2+ in myofibrils isolated from stunned and nonstunned porcine and human myocardium.

Previously, we showed, in an in situ porcine model, that the thiadiazinone derivative [+]EMD 60263, a putative Ca2+ sensitizer with minimal phosphodiesterase III inhibitory properties, increased contractility more profoundly in stunned than in nonstunned myocardium. The aim of the present investigation was to study the mechanism of action by determining the in vitro effects of [+]EMD 60263 on the Ca2+ responsiveness of the Mg(2+)-dependent ATPases of myofibrils and sarcoplasmic reticulum membrane vesicles, isolated from normal ventricle of swine and hypertrophic septum of cardiomyopathic patients. Contamination of the myofibrils with sarcoplasmic reticulum membranes was excluded by testing the effect of the sarcoplasmic reticulum Ca(2+)-pumping ATPase inhibitor thapsigargin. The plasma concentrations at which [+]EMD 60263 exerted its inotropic effect in the in situ porcine model were found to be submicromolar. [+]EMD 60263 stimulated concentration-dependently (1-10 microM) the submaximally activated Mg(2+)-ATPases (at pCa 6.1) of pig heart myofibrils. [+]EMD 60263 (10 microM) shifted the pCa50 of porcine myofibrillar Ca(2+)-stimulated, Mg(2+)-dependent ATPase from 6.00 +/- 0.05 to 6.67 +/- 0.05, whereas the [-]enantiomer EMD 60264 had no significant effect. Although the effect was much less at 1 and 3 microM, [+]EMD 60263 (10 microM) also stimulated maximal myofibrillar Mg(2+)-ATPase activity. The Hill coefficient, reflecting the steepness of the fitted pCa/Mg(2+)-ATPase curve at half-maximal activation, was not affected by [+]EMD 60263 (10 microM). [+]EMD 60263 (10 microM) had no effect on sarcoplasmic reticulum Ca(2+)-stimulated, Mg(2+)-dependent ATPase from swine heart. The thiadiazinone derivative [+]EMD 57033 (10 microM), but not its [-]enantiomer EMD 57439, had similar, although less potent, effects on pig heart myofibrillar Mg(2+)-ATPase activity as compared to [+]EMD 60263. [+]EMD 60263 (3 microM) produced a significantly larger leftward shift of the pCa2+/Mg(2+)-ATPase activity curve of myofibrils isolated from the stunned compared to the adjacent nonstunned myocardium (Delta pCa50s caused by the presence of [+]EMD 60263 amounted to +0.57 +/- 0.04 and +0.42 +/- 0.05, respectively) in the in situ porcine model. The effects of [+]EMD 60263 on myofibrillar Mg(2+)-ATPase of hypertrophic human heart were identical to those observed with porcine heart myofibrils. The results indicate that the positive inotropic action of [+]EMD 60263 observed in the in situ porcine model of stunned myocardium may be primarily due to myofilament sensitization to Ca2+, and that this compound may have a similar action on diseased human myocardium.

In vitro analysis of SERCA2 gene regulation in hypertrophic cardiomyocytes and increasing transfection efficiency by gene-gun biolistics.

The transcriptional downregulation of the SERCA2 gene is studied using neonatal rat cardiomyocytes stimulated with endothelin-1 to induce hypertrophy. Liposome-based transfection of cells with a 1.9 kb SERCA2 promoter fragment directed expression of a reporter gene identical to the downregulation of genomic SERCA2 expression by endothelin-1. Results of a new gene gun technology for transient transfection of cardiomyocytes with a RSV-beta-galactosidase construct are reported. This new method for propelling DNA-coated gold beads into cardiomyocytes is extremely suitable for directly testing promoter/reporter gene DNA constructs since the transfection efficiency (approximately 10%) appears to be higher than traditional transfection methods.

Identification by a differential proteomic approach of the induced stress and redox proteins by resveratrol in the normal and diabetic rat heart.

A recent study showed cardioprotective effects of resveratrol on the diabetic heart. The present study sought to compare the protein profiles of the normal versus diabetic hearts after resveratrol treatment using differential proteomic analysis. Rats were randomly divided into two groups: control and diabetic. Both groups of rats were fed resveratrol (2.5 mg/kg/day) for 7 days, and then the rats were sacrificed, hearts were isolated and cytoplasmic fraction from left ventricular tissue was collected to carry out proteomic profiling as well as immunoblotting. Compared to normal hearts, diabetic hearts show increased myocardial infarct size and cardiomy-ocyte apoptosis upon ex vivo global ischaemia of 30 min. followed by 2 hrs of reperfusion. Resveratrol reduced infarct size and apop-totic cell death for both the groups, but the extent of infarct size and apoptosis remained higher for the diabetic group compared to the normal group. The left ventricular cytoplasmic proteins were analysed by 2D-DIGE and differentially displayed bands were further analysed by nano Liquid Chromatography-Mass Spectroscopy (LC-MS/MS). The results showed differential regulation of normal versus diabetic hearts treated with resveratrol of many proteins related to energy metabolism of which several were identified as mitochondrial proteins. Of particular interest is the increased expression of several chaperone proteins and oxidative stress and redox proteins in the diabetic group including Hsc70, HSPp6, GRP75, peroxiredoxin (Prdx)-1 and Prdx-3 whose expression was reversed by resveratrol. Western blot analysis was performed to validate the up- or down-regulation of these stress proteins. The results indicate the differential regulation by resveratrol of stress proteins in diabetic versus normal hearts, which may explain in part the beneficial effects of resveratrol in diabetic induced cardiovascular complications.

Endothelin-1 and phenylephrine-induced activation of the phosphoinositide cycle increases cell injury of cultured cardiomyocytes exposed to hypoxia/reoxygenation.

We explored the effect of glucose-free hypoxia/reoxygenation of cultured neonatal rat ventricular myocytes on endothelin-1 and alpha 1-adrenoceptor induced activity of the phosphoinositide cycle. At the same time the influence of these agonists on depletion of energy-rich phosphates and cellular damage was assessed. Glucose-free hypoxia did not lead to an increase in basal phospholipase C activity. However, endothelin-1 (10(-8) M) and phenylephrine (10(-5) M) evoked activation of phospholipase C was attenuated after 60 min of hypoxia and declined to 38% and 30% respectively of normoxic values after 90 min of hypoxia. During glucose-free hypoxia, phosphatidylinositol 4,5-bisphosphate, the substrate for phospholipase C, but not phosphatidylinositol or phosphatidylinositol 4-monophosphate was seen to decline to 59% of normoxic values which was independent of activation of phospholipase C by agonists. ATP levels decreased after 30 min of hypoxia and declined to 29% relative to normoxic control after 90 min of hypoxia. Total adenine nucleotide levels showed a similar pattern. The presence of 10(-8) M endothelin-1 during hypoxia did not influence the magnitude of ATP depletion. However, after 15 min of reoxygenation, by itself not significantly leading to recovery of ATP levels, ATP levels were decreased by endothelin-1 as compared to hypoxia/reoxygenation without phospholipase C agonist. Cellular damage as determined by lactate dehydrogenase leakage was not observed during 90 min hypoxia. Reoxygenation resulted in a three-fold increase in enzyme release relative to normoxic control. In the presence of endothelin-1 or phenylephrine this reoxygenation-induced damage was respectively 1.7 and 3.0-fold increased. We conclude that the agonist-induced activity of the phosphoinositide cycle is decreased in time during glucose-free hypoxia, partially through a decrease in phosphatidylinositol 4,5-bisphosphate level. However, the remaining activity may give rise to increased cellular damage. As endothelin-1 and alpha 1-adrenergic amines are known to be released during myocardial ischemia, stimulation of the phosphoinositide cycle by these agonists might be an important factor in determining the magnitude of myocardial injury.

Phorbol ester and the actions of phosphatidylinositol 4,5-bisphosphate specific phospholipase C and protein kinase C in microsomes prepared from cultured cardiomyocytes.

Microsomes were prepared from cultured neonatal rat cardiomyocytes. Incubation of microsomes in buffer containing 5 microM CaCl2, 5 mM cholate and 100 nM [3H-]Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5) P2) resulted in the formation of [3H-]InsP3. GTP-gamma-S (125 microM) stimulated the production of [3H-]InsP3. Microsomes prepared from phorbol ester-treated (100 nM phorbol 12-myristate 13-acetate, PMA) cardiomyocytes showed decreased activities of basal as well as GTP-gamma-S-stimulated [3H-]PtdIns(4,5)P2 hydrolysis. In the microsomes a 15 kD protein was demonstrated to be the major substrate phosphorylated by intrinsic protein kinase C, which was activated by 0.5 mM Ca2+. Addition of phorbol ester (100 nM PMA) enhanced the 32P-incorporation into the 15 kD protein. Protein kinase C, purified from rat brain, in the presence of Ca2+, diglyceride, and phosphatidylserine did not change the phosphorylation pattern any further. In conclusion, it was shown that phorbol ester pretreatment of neonatal rat cardiomyocytes reduces microsomal GTP-gamma-S-stimulated PtdIns(4,5)P2-specific phospholipase C activity, as estimated with exogenous substrate, and that in cardiomyocyte microsomes phorbol ester activates protein kinase C-induced 15 kD protein phosphorylation. The results indicate that phorbol ester may down-regulate alpha 1-adrenoceptor mediated PtdIns(4,5)P2 hydrolysis by activation of protein kinase C-induced 15 kD protein phosphorylation.

Homologous desensitization of the endothelin-1 receptor mediated phosphoinositide response in cultured neonatal rat cardiomyocytes.

The goal of the present study was to identify the molecular mechanism underlying desensitization of endothelin-1 receptor-mediated phosphoinositide response in cultured neonatal rat heart cells. Endothelin elicited a concentration-dependent (EC50 = 2.2 x 10(-9) M) increase of inositol-phosphate production with a much higher potency than phenylephrine (EC50 = 1.4 x 10(-6) M). Endothelin-1 (10(-8) M) evoked phosphoinositide turnover in the presence of 10 mM LiCl, which was greatly attenuated after 30-45 min of continuous stimulation with agonist, apparently resulting in a total absence of further inositol-phosphate accumulation. However, when the uncompetitive inositol monophosphatase inhibitor Li+ was only present during the last 30 min of 150 min incubation, the inositol-phosphate accumulation was decreased to a steady state of 33% of the initial rate. The loss of responsiveness of cardiomyocytes to endothelin-1 was not brought about by a limiting supply of phospholipase C substrate phosphatidylinositol 4,5-bisphosphate. A very rapid resynthesis of this substrate took place as its level remained almost constant during 45 min stimulation with 10(-8) M endothelin-1 while the accumulation of inositol-phosphates was at least 15-fold higher than the initial cellular phosphatidylinositol 4,5-bisphosphate content. After 120 min preincubation of cells with 10(-9) M endothelin-1 the activation of phospholipase C by a second higher dose (10(-8) M) was severely (67%) inhibited at the same time leaving the induction of phosphoinositide turnover by phenylephrine (10(-4) M) virtually intact. Preincubation with phenylephrine (3 x 10(-6) M) also led to inhibition of the phenylephrine (10(-4) M)-mediated inositol-phosphate response (36% inhibition) while the endothelin-1 (10(-8) M) response was not affected. Addition of a direct activator of protein kinase C, phorbol 12-myristate 13-acetate, led to inhibition of the endothelin-1 evoked phosphoinositide turnover but the rate of desensitization was not affected. Inhibition of protein kinase C with staurosporine did not alter the time course of desensitization. In conclusion, the activity of the phosphoinositide cycle in cardiomyocytes is homologously desensitized after stimulation with endothelin-1. The desensitization is not likely to be due to either depletion of phospholipase C substrate or to the activation of protein kinase C by inositol 1,4,5-trisphosphate-mobilized Ca2+ and elevated 1,2-diacylglycerol levels.

Calcium and the endothelin-1 and alpha 1-adrenergic stimulated phosphatidylinositol cycle in cultured rat cardiomyocytes.

Cultured neonatal rat cardiac myocytes have been utilized as a model for the study of the effect of variations in cytoplasmic free Ca2+ on the activity of phospholipase C, a key enzyme in agonist-stimulated signal transduction through the phosphoinositide pathway. Cells prelabelled with [3H]inositol were exposed to various agents in an attempt to modulate the cytoplasmic free Ca2+ concentration and the formation of [3H]inositolphosphates (15-30 min) in the presence of Li+ was taken as a measure of phospholipase C activity. Not the basal but the endothelin-1 (10(-8) M) induced [3H]inositolphosphate production (15 min) was stimulated 1.54- and 1.43-fold by A23187 (10 microM external Ca2+) and 50 mM K+ (1.3 mM external Ca2+) treatment of cells, respectively. The phenylephrine (10(-4) M) induced response was also stimulated (1.35-fold) by A23187, however it was 43% inhibited by high K+. Ouabain (10 microM) treatment of cells did not affect either basal or agonist stimulated phosphoinositide turnover. On the other hand, total removal of external free Ca2+ by addition of 50 microM ethylene glycol bis(beta-aminoethyl ether) (N,N,N',N'-tetraacetic acid strongly inhibited (75%) the endothelin-1 induced but not the basal phospholipase C activity. Endothelin-1 binding to its receptor was shown not to be inhibited by the absence of external Ca2+ while resynthesis of [3H]phosphatidylinositol 4,5-bisphosphate was not rate-limiting under this condition. The lack of external Ca2+ eventually resulted in total standstill of the ET-1 induced PtdIns turnover after 30 min. Although not always as predicted, effects on basal and agonist-activated phospholipase C were observed too when cells were treated with low Ca2+ medium, Ca2+ entry blocker nifedipine (1 microM) or Ca(2+)-channel agonist Bay K8644 (1 microM) but most of these effects were only seen after 90 min incubation. Fluorometric (fura-2) measurements showed that total removal of external free Ca2+ for a short period decreased, while short exposure to high K+ increased cytoplasmic free Ca2+ but neither Ca2+ free buffer or nifedipine nor Bay K8644 had any effect. Furthermore, in saponin-permeabilized cardiomyocytes we could demonstrate that basal as well as GTP gamma S (30 microM) stimulated phospholipase C activity was strongly activated by free Ca2+ in the concentration range of 0.1-10 microM. We conclude that in the intact cardiomyocyte the signalling pathway through phospholipase C/phosphatidylinositol 4,5-bisphosphate, stimulated by agonist-receptor interaction that activates GTP-binding proteins as does GTP gamma S, is likely be a Ca2+ dependent process.

Distinct alpha 1-adrenergic agonist- and endothelin-1-evoked phosphoinositide cycle responses in cultured neonatal rat cardiomyocytes.

Previously it was shown by us and others in cultured neonatal rat cardiomyocytes that the desensitization of the phenylephrine (PHE)- and endothelin-1 (ET-1)-mediated response of phospholipase C (PLC) was receptor-specific. The aim of this study was to characterize receptor-dependent specificities downstream of PLC. PHE (10(-4)M) as well as ET-1 (10(-8) M) stimulated the total [3H]inositolphosphate ([3H]InsPn, predominantly [3H]Ins(4)P) formation to about the same extent whereas Ins(1,4,5)P3 and Ins(1,3,4,5)P4 did not increase. Yet, ET-1 but not PHE stimulated Ins(1,3,4)P3 and Ins(3,4)Pn formation. Activation of PLC in saponin-permeabilized cells by GTP gamma S- and Ca2+ gave predominantly the formation of Ins(1,4)P2. The PHE- and ET-1-mediated increase of [3H]1,2-diacylglycerol was significant after respectively 16 and 8 min. PHE but not ET-1 stimulated phosphorylation of a 30 kDa protein which was likely of myofibrillar origin. It is concluded that receptor-dependent specificities exist not only at the level of PLC but also downstream.

The influence of the lipid composition on the degree of lipid-peroxidation of liposomes.

Liposomes containing dilinoleoyl lecithin (di-18(2)PC) and diarachidonyl lecithin (di-20(4)PC) were peroxidized with Fe++/ascorbate. Lipidperoxidation was measured as generated malondialdehyde, the disappearance of polyunsaturated fatty acids, conjugated diene formation and chemiluminescense. It was found that di-20(4)PC was oxidized rapidly, while in the same time period hardly any oxidation of di-18(2)PC took place. Incorporation of different lipid classes into the di-20(4)PC liposomes influenced the peroxidation for each lipid in a characteristic way. Phosphatidylethanolamine stimulated the formation of fluorescent chromolipids. The possible meaning of these reactions in human pathology is discussed.

Phosphorylation by protein kinase C and the responsiveness of Mg(2+)-ATPase to Ca2+ of myofibrils isolated from stunned and non-stunned porcine myocardium.

Previously we showed in an in situ porcine model that the thiadiazinone derivative [+]EMD 60263, a Ca2+ sensitizer without phosphodiesterase III inhibitory properties, increased contractility more profoundly in stunned than in non-stunned myocardium. This finding was consistent with the observed leftward shifts of the pCa2+/Mg(2+)-ATPase curves of isolated myofibrils induced by [+]EMD 60263. The aim of the present investigation was to study the possible involvement of protein kinase C in the mechanism of reduced Ca2+ responsiveness of myofilaments during stunning. No differences were observed in the maximal activity of the Ca(2+)-stimulated Mg(2+)-ATPase and in the pCa50 of myofibrils isolated from non-stunned and stunned myocardium. After phosphorylation with [gamma-32P]-ATP and excess of purified rat brain protein kinase C, the myofibrils were separated on sodiumdodecylsulphate-polyacrylamide gelectrophoresis and the 32P incorporation counted by the Molecular Imager. Ca2+/ phosphatidylserine/sn-1,2 diolein-dependent 32P incorporation catalyzed by excess of purified rat brain protein kinase C in C-protein, TnT and TnI subunits did not show any differences between myofibrils from non-stunned and stunned myocardium. However, protein kinase C-induced phosphorylation of myofibrils isolated from ventricular myocardium of sham-operated pigs resulted in a marked leftward shift of the pCa50 from 6.03 +/- 0.04 to 6.44 +/- 0.06 (p < 0.05), while porcine heart cyclic AMP-dependent protein kinase-induced phosphorylation resulted in an expected small rightward shift to 5.97, although statistical significance was not reached. Protein kinase C-induced phosphorylation also stimulated (80%) the maximal myofibrillar Mg(2+)-ATPase activity. [+]EMD 60263 (3 microM) produced a leftward shift of the myofibrillar pCa2+/Mg(2+)-ATPase curve which was unaffected by prior protein kinase C-induced phosphorylation. In conclusion, the findings with isolated myofibrils from myocardium of anaesthetized open-chest pigs indicate that protein kinase C might be involved in the mechanism of reduced Ca2+ responsiveness of myofilaments in stunned myocardium. However, at this stage no differences could be found between the maximal activity of the Ca(2+)-stimulated Mg(2+)-ATPase, the pCa50 and the degree of phosphorylation of myofibrils isolated from stunned and non-stunned myocardium.

Negative inotropy of lidocaine: possible biochemical mechanisms.

Previous studies have shown that lidocaine has a negative inotropic effect on the myocardium. This effect could be mediated by a decrease in O2 supply and/or utilization, or abnormalities in intracellular Ca2+ handling by the myocardium. To investigate which of these mechanisms are involved we studied nine open-chest anaesthetized pigs, which received an infusion of lidocaine (4-16 mg.min-1) in the left anterior descending coronary artery (LADCA), sufficient to induce a severe depression of the regional myocardial function. Biopsies for high energy phosphate levels were obtained from both the LADCA and control regions before and during the infusion. After measurements at peak lidocaine dose, the hearts were rapidly excised for harvesting of LADCA, and control region sarcoplasmic reticulum (SR) vesicles for in vitro measurements of Ca2+ uptake rate. During lidocaine infusion, coronary blood flow increased (23%), while ATP, Ca2+ uptake by the SR and percentage segment length shortening decreased by 20%, 19% and 30% respectively. However, O2 consumption in the LADCA region did not differ before or during lidocaine infusion (102 +/- 20 and 104 +/- 29 ml.min-1, respectively). Hence, lidocaine in doses sufficient to depress regional myocardial function does not decrease O2 supply, but decreases the efficiency of oxygen utilization. Although we cannot entirely rule out the possibility that blockade of fast sodium channels is a contributory factor, the observed decrease in the tissue level of ATP and the rate of Ca2+ uptake by the SR may be related to the negative inotropic action of lidocaine.(ABSTRACT TRUNCATED AT 250 WORDS)