An interaction network of mental disorder proteins in neural stem cells.

Mental disorders (MDs) such as intellectual disability (ID), autism spectrum disorders (ASD) and schizophrenia have a strong genetic component. Recently, many gene mutations associated with ID, ASD or schizophrenia have been identified by high-throughput sequencing. A substantial fraction of these mutations are in genes encoding transcriptional regulators. Transcriptional regulators associated with different MDs but acting in the same gene regulatory network provide information on the molecular relation between MDs. Physical interaction between transcriptional regulators is a strong predictor for their cooperation in gene regulation. Here, we biochemically purified transcriptional regulators from neural stem cells, identified their interaction partners by mass spectrometry and assembled a protein interaction network containing 206 proteins, including 68 proteins mutated in MD patients and 52 proteins significantly lacking coding variation in humans. Our network shows molecular connections between established MD proteins and provides a discovery tool for novel MD genes. Network proteins preferentially co-localize on the genome and cooperate in disease-relevant gene regulation. Our results suggest that the observed transcriptional regulators associated with ID, ASD or schizophrenia are part of a transcriptional network in neural stem cells. We find that more severe mutations in network proteins are associated with MDs that include lower intelligence quotient (IQ), suggesting that the level of disruption of a shared transcriptional network correlates with cognitive dysfunction.

Detection of Alpha-Toxin and Other Virulence Factors in Biofilms of Staphylococcus aureus on Polystyrene and a Human Epidermal Model.

BACKGROUND & AIM: The ability of Staphylococcus aureus to successfully colonize (a)biotic surfaces may be explained by biofilm formation and the actions of virulence factors. The aim of the present study was to establish the presence of 52 proteins, including virulence factors such as alpha-toxin, during biofilm formation of five different (methicillin resistant) S. aureus strains on Leiden human epidermal models (LEMs) and polystyrene surfaces (PS) using a competitive Luminex-based assay. RESULTS: All five S. aureus strains formed biofilms on PS, whereas only three out of five strains formed biofilms on LEMs. Out of the 52 tested proteins, six functionally diverse proteins (ClfB, glucosaminidase, IsdA, IsaA, SACOL0688 and nuclease) were detected in biofilms of all strains on both PS and LEMs. At the same time, four toxins (alpha-toxin, gamma-hemolysin B and leukocidins D and E), two immune modulators (formyl peptide receptor-like inhibitory protein and Staphylococcal superantigen-like protein 1), and two other proteins (lipase and LytM) were detectable in biofilms by all five S. aureus strains on LEMs, but not on PS. In contrast, fibronectin-binding protein B (FnbpB) was detectable in biofilms by all S. aureus biofilms on PS, but not on LEMs. These data were largely confirmed by the results from proteomic and transcriptomic analyses and in case of alpha-toxin additionally by GFP-reporter technology. CONCLUSION: Functionally diverse virulence factors of (methicillin-resistant) S. aureus are present during biofilm formation on LEMs and PS. These results could aid in identifying novel targets for future treatment strategies against biofilm-associated infections.

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.

Alterations in myofilament function contribute to left ventricular dysfunction in pigs early after myocardial infarction.

Myocardial infarction (MI) initiates cardiac remodeling, depresses pump function, and predisposes to heart failure. This study was designed to identify early alterations in Ca2+ handling and myofilament proteins, which may contribute to contractile dysfunction and reduced beta-adrenergic responsiveness in postinfarct remodeled myocardium. Protein composition and contractile function of skinned cardiomyocytes were studied in remote, noninfarcted left ventricular (LV) subendocardium from pigs 3 weeks after MI caused by permanent left circumflex artery (LCx) ligation and in sham-operated pigs. LCx ligation induced a 19% increase in LV weight, a 69% increase in LV end-diastolic area, and a decrease in ejection fraction from 54+/-5% to 35+/-4% (all P<0.05), whereas cardiac responsiveness to exercise-induced increases in circulating noradrenaline levels was blunted. Endogenous protein kinase A (PKA) was significantly reduced in remote myocardium of MI animals, and a negative correlation (R=0.62; P<0.05) was found between cAMP levels and LV weight-to-body weight ratio. Furthermore, SERCA2a expression was 23% lower after MI compared with sham. Maximal isometric force generated by isolated skinned myocytes was significantly lower after MI than in sham (15.4+/-1.5 versus 19.2+/-0.9 kN/m2; P<0.05), which might be attributable to a small degree of troponin I (TnI) degradation observed in remodeled postinfarct myocardium. An increase in Ca2+ sensitivity of force (pCa50) was observed after MI compared with sham (DeltapCa50=0.17), which was abolished by incubating myocytes with exogenous PKA, indicating that the increased Ca2+ sensitivity resulted from reduced TnI phosphorylation. In conclusion, remodeling of noninfarcted pig myocardium is associated with decreased SERCA2a and myofilament function, which may contribute to depressed LV function. The full text of this article is available online at http://circres.ahajournals.org.

High-affinity prorenin binding to cardiac man-6-P/IGF-II receptors precedes proteolytic activation to renin.

Mannose-6-phosphate (man-6-P)/insulin-like growth factor-II (man-6-P/IgF-II) receptors are involved in the activation of recombinant human prorenin by cardiomyocytes. To investigate the kinetics of this process, the nature of activation, the existence of other prorenin receptors, and binding of native prorenin, neonatal rat cardiomyocytes were incubated with recombinant, renal, or amniotic fluid prorenin with or without man-6-P. Intact and activated prorenin were measured in cell lysates with prosegment- and renin-specific antibodies, respectively. The dissociation constant (K(d)) and maximum number of binding sites (B(max)) for prorenin binding to man-6-P/IGF-II receptors were 0.6 +/- 0.1 nM and 3,840 +/- 510 receptors/myocyte, respectively. The capacity for prorenin internalization was greater than 10 times B(max). Levels of internalized intact prorenin decreased rapidly (half-life = 5 +/- 3 min) indicating proteolytic prosegment removal. Prorenin subdivision into man-6-P-free and man-6-P-containing fractions revealed that only the latter was bound. Cells also bound and activated renal but not amniotic fluid prorenin. We concluded that cardiomyocytes display high-affinity binding of renal but not extrarenal prorenin exclusively via man-6-P/IGF-II receptors. Binding precedes internalization and proteolytic activation to renin thereby supporting the concept of cardiac angiotensin formation by renal prorenin.

Cultured neonatal rat cardiac myocytes and fibroblasts do not synthesize renin or angiotensinogen: evidence for stretch-induced cardiomyocyte hypertrophy independent of angiotensin II.

OBJECTIVE: The hypertrophic response of cardiomyocytes exposed to mechanical stretch is assumed to depend on the release of angiotensin (Ang) II from these cells. Here we studied the synthesis of renin-angiotensin system (RAS) components by cardiac cells under basal conditions and after stretch. METHODS: Myocytes and fibroblasts were isolated by enzymatic dissociation from hearts of 1-3-day-old Wistar rat strain pups, grown for 1 day in serum-supplemented medium and then cultured in a chemically defined, serum-free medium. Medium and cell lysate were collected 5 days later or after exposure of the cells to cyclic stretch for 24 h. Prorenin, renin and angiotensinogen were measured by enzyme-kinetic assay; Ang I and Ang II were measured by radioimmunoassay after SepPak extraction and HPLC separation. RESULTS: Prorenin, but none of the other RAS components, could be detected in the medium of both cell types. However, its levels were low and the Ang I-generating activity corresponding with these low prorenin levels could not be inhibited by the specific rat renin inhibitor CH-732, suggesting that it was most likely due to bovine and/or horse prorenin sequestered from the serum-containing medium to which the cells had been exposed prior to the serum-free period. When incubated with Ang I, both myocytes and fibroblasts generated Ang II in a captopril-inhibitable manner. Myocyte and fibroblast cell lysates did not contain prorenin, renin, angiotensinogen, Ang I or Ang II in detectable quantities. Stretch increased myocyte protein synthesis by 20%, but was not accompanied by Ang II release into the medium. CONCLUSION: Cardiac myocytes and fibroblasts do not synthesize renin, prorenin or angiotensinogen in concentrations that are detectable or, it not detectable, high enough to result in Ang II concentrations of physiological relevance. These cells do synthesize ACE, thereby allowing the synthesis of Ang II at cardiac tissue sites when renin and angiotensinogen are provided via the circulation. Ang II is not a prerequisite to observe a hypertrophic response of cardiomyocytes following stretch.

Phospholipid source and molecular species composition of 1,2-diacylglycerol in agonist-stimulated rat cardiomyocytes.

OBJECTIVE: The aim was to investigate the consequences of simultaneous stimulation of phospholipase C and D by agonists for the molecular species composition of 1,2-diacylglycerol and phospholipids in cardiomyocytes. METHODS: Serum-free cultured neonatal rat cardiomyocytes were stimulated by endothelin-1, phenylephrine or phorbolester. The molecular species of 1,2-diacylglycerol (in mol%) and those derived from phosphatidylcholine and phosphatidylinositol were analyzed by high-performance liquid chromatography and their absolute total concentration (nmol per dish) by gas-liquid chromatography. Phospholipids were labelled with [14C]glycerol or double-labelled with [14C]16:0 and [3H]20:4n6 for measurements of respectively, the amount of or relative rate of label incorporation into 1,2-diacylglycerol. RESULTS: The major molecular species of 1,2-diacylglycerol in unstimulated cells was found to be 18:0/20:4 (57 mol%). The same species was observed predominantly in phosphatidylinositol (73 mol% compared to 11 mol% in phosphatidylcholine). A significant decrease (about 10 mol%) was found for the 18:0/20:4 species of 1,2-diacylglycerol during stimulation (10-40 min) with endothelin-1 or phorbolester, but not phenylephrine. The results of the double-labelling experiments were consistent with the latter finding: the ratio [3H]20:4 over [14C]16:0 in 1,2-diacylglycerol decreased from 1.70 in the control to 1.40 during 10-min endothelin-1 or phorbolester stimulation, but not during phenylephrine stimulation. The [14C]glycerol incorporation into 1,2-diacylglycerol remained relatively constant under agonist-stimulated conditions as did the total concentration of 1,2-diacylglycerol. CONCLUSIONS: 1,2-Diacylglycerol present in unstimulated cardiomyocytes is likely derived from phosphatidylinositol. During stimulation with endothelin-1 and phorbolester, but not phenylephrine, phosphatidylcholine becomes an increasingly important source for 1,2-diacylglycerol due to sustained activation of phospholipase D. The 1,2-diacylglycerol level remains relatively constant during agonist stimulation which strongly indicates that particular molecular species of 1,2-diacylglycerol more than its total concentration determine the activation of protein kinase C isoenzymes.

Mannose 6-phosphate receptor-mediated internalization and activation of prorenin by cardiac cells.

The binding and internalization of recombinant human renin and prorenin (2500 microU/mL) and the activation of prorenin were studied in neonatal rat cardiac myocytes and fibroblasts cultured in a chemically defined medium. Surface-bound and internalized enzymes were distinguished by the addition of mannose 6-phosphate to the medium, by incubating the cells both at 37 degrees C and 4 degrees C, and by the acid-wash method. Mannose 6-phosphate inhibited the binding of renin and prorenin to the myocyte cell surface in a dose-dependent manner. At 37 degrees C, after incubation at 4 degrees C for 2 hours, 60% to 70% of cell surface-bound renin or prorenin was internalized within 5 minutes. Intracellular prorenin was activated, but extracellular prorenin was not. The half-time of activation at 37 degrees C was 25 minutes. Ammonium chloride and monensin, which interfere with the normal trafficking and recycling of internalized receptors and ligands, inhibited the activation of prorenin. Results obtained with cardiac fibroblasts were comparable to those in the myocytes. This study is the first to show experimental evidence for the internalization and activation of prorenin in extrarenal cells by a mannose 6-phosphate receptor-dependent process. Our findings may have physiological significance in light of recent experimental data indicating that angiotensin I and II are produced at cardiac and other extrarenal tissue sites by the action of renal renin and that intracellular angiotensin II can elicit important physiological responses.

Cross-talk between receptor-mediated phospholipase C-beta and D via protein kinase C as intracellular signal possibly leading to hypertrophy in serum-free cultured cardiomyocytes.

Phospholipase C-beta (PLC-beta) signalling via protein kinase C (PKC) has been recognized as a major route by which stimuli such as alpha1-adrenergic agonists, endothelin-1 (ET-1) and angiotensin II (Ang II) induce hypertrophy of myocytes. The goal of this study was to evaluate the role of phospholipase D (PLD) in contributing to the formation of the PKC activator 1,2-diacylglycerol (1,2-DAG) and to study the mechanism(s) of PLD activation by agonists. Stimulation of serum-free cultured neonatal rat cardiomyocytes with ET-1 (10(-8)M), phenylephrine (PHE, 10(-5)M) or Ang II (10(-7)M) resulted in a rapid (0-10 min) activation of PLC-beta to an extent (ET-1>PHE>Ang II) that correlated with the magnitude of stimulation of protein synthesis ([3H]leucine incorporation into protein) measured after 24 h. Phorbol 12-myristate 13-acetate (PMA, 10(-6)M) and ET-1 were equipotent in stimulating protein synthesis. ET-1 and PMA, but not PHE and Ang II stimulated [3H]choline formation from labelled PtdCho after a lag-phase of about 10 min. That this [3H]choline formation was due to the action of PLD was confirmed by measurement of phosphatidylgroup-transfer from cellular [14C]palmitoyl-phosphatidylcholine to exogenous ethanol. ET-1 and PHE, to much lesser extent, produced a rapid (0-5 min) translocation of PKC- immunoreactivity from the cytosol to the membrane fraction, whereas no intracellular redistribution of PKC-alpha, -delta and -xi immunoreactivities was observed. PMA caused translocation of PKC-alpha, PKC-epsilon as well as PKC-delta. Cellular redistribution of PKC activity measured by [32P]-incorporation into histone III-S was not observed with ET-1 and PHE, but only with PMA stimulation. Down-regulation of PKC isozymes by 24 h pretreatment of cells with PMA or blockade of PKC by chelerythrine (10(-4)M) inhibited ET-1 and PMA stimulated [3H]choline production. Staurosporine (10(-6)M) had, however, no effect. In conclusion, the results indicate that in serum-free cultured cardiomyocytes, ET-1 initially activates PLC-beta and after a lag-phase PLD, whereas PHE and Ang II activate only PLC-beta. PLC-beta stimulated by ET-1, may cross-talk with PLD via translocation of PKC-epsilon. These signals are possibly linked to the hypertrophic response.

Eicosapentaenoic acid incorporation in membrane phospholipids modulates receptor-mediated phospholipase C and membrane fluidity in rat ventricular myocytes in culture.

The influence of increased incorporation of linoleic acid (18:2n-6) and eicosapentaenoic acid (20:5n-3) in membrane phospholipids on receptor-mediated phospholipase C beta (PLC-beta) activity in cultured rat ventricular myocytes was investigated. For this purpose, cells were grown for 4 days in control, stearic acid (18:0)/oleic acid (18:1n-9), 18:2n-6 and 20:5n-3 enriched media, and subsequently assayed for the basal- and phenylephrine- or endothelin-1-induced total inositol phosphate formation. The various fatty acid treatments resulted in the expected alterations of fatty acid composition of membrane phospholipids. In 18:2n-6-treated cells, the incorporation of this 18:2n-6 in the phospholipids increased from 17.1 mol % in control cells to 38.9 mol %. In 20:5n-3-treated cells, incorporation of 20:5n-3 and docosapentaenoic acid (22:5n-3) in the phospholipids increased from 0.5 and 2.7 mol % in control cells to 23.2 and 9.7 mol %, respectively. When 20:5n-3-treated cells were stimulated with phenylephrine or endothelin-1, the inositolphosphate production decreased by 33.2% and increased by 43.4%, respectively, as compared to cells grown in control medium. No effects were seen in 18:2n-6-treated cells. When 18:0/18:1n-9-treated cells were stimulated with endothelin-1, inositolphosphate formation increased by 26.4%, whereas phenylephrine-stimulated inositolphosphate formation was not affected. In saponin-permeabilized cells, that were pre-treated with 20:5n-3, the formation of total inositolphosphates after stimulation with GTP gamma S, in the presence of Ca2+, was inhibited 19.3%. This suggests that the 20:5n-3 effect on intact cardiomyocytes could be exerted either on the level of agonist-receptor, receptor-GTP-binding-protein coupling or GTP-binding-protein-PLC-beta interaction. Investigation of the time course of saponin-induced permeabilization of the cardiomyocytes, measured by the release of lactate dehydrogenase, unmasked a slight decrease in the rate of permeabilization by 20:5n-3 pretreatment, indicating a protective effect. This led the authors to measure the cholesterol/phospholipid molar ratio, the double bond index of membrane phospholipids, and the membrane fluidity; the latter by using a diphenylhexatriene probe. In 20:5n-3-pretreated cells, a strong increase in the cholesterol/phospholipid molar ratio (from 0.23 to 0.39), a marked increase in the double bond index (from 1.76 to 2.33), and a slight decrease in fluidity (steady-state anisotropy rss of the diphenylhexatriene probe increased from 0.196 to 0.217) were observed. Thus, treatment of cardiomyocytes for 4 days with 20:5n-3, but not with 18:2n-6, causes alterations of receptor-mediated phospholipase C beta activity. A causal relationship may exist between the 20:5n-3 causes alterations of the physicochemical properties in the bilayer and of the agonist-stimulated phosphatidylinositol cycle activity.

Polyunsaturated fatty acids and signalling via phospholipase C-beta and A2 in myocardium.

Dietary n-6 and n-3 polyunsaturated fatty acids (PUFAs) have potent biological effects on the blood(cells), the vasculature and they myocardium. In the epidemiological studies in which the benefit from the regular ingestion of n-3 PUFAs was reported, the responsible mechanisms remain obscure. A great deal of the PUFA-effect can be explained by the known interference with the eicosanoid metabolism. Many processes, believed to be involved in atherogenesis such as adhesion and infiltration of bloodcells (in)to the vasculature, platelet aggregation, secretion of endothelium-derived factors and mitogenic responses of vascular smooth muscle cells are partially mediated by receptor-activated phospholipases C-beta and A2. As PUFAs take part at many steps of the signalling pathways, the latter could represent important action sites to beneficially interfere with atherogenesis. In this brief review, we have discussed the results of studies on the influence of alteration of PUFA composition of the membrane phospholipids or of exogenously administered non-esterified PURAs on phospholipid signalling. For convenience, we have mainly focused our discussion on those studies available on the myocardium. By changing the PUFA composition of the phospholipids, the endogenous substrates for the membrane-associated phospholipase C-beta and A2 are changed. This is accompanied by changes in their hydrolytic action on these substrates resulting in altered products (the molecular species of 1,2-diacylglycerols and the non-esterified PUFAs) which on their turn evoke changes in events downstream of the signalling cascades: activation of distinct protein kinase C isoenzymes, formation of distinct eicosanoids and non-esterified PUFA effects on Ca2+ channels. It has also become more clear that the membrane physicochemical properties, in terms of fluidity and cholesterol content of the bilayer, might undergo changes due to altered PUFA incorporation into the membrane phospholipids. The latter effects could have consequences for the receptor functioning, receptor-GTP-binding protein coupling, GTP-binding protein-phospholipase C-beta or A2 coupling as well. It should be noted that most of these studies have been carried out with cardiomyocytes isolated from hearts of animals on PUFA diet or incubation of cultured cardiomyocytes with non-esterified PUFAs in the presence of albumin. Studies need to be performed to prove that the PUFA-diet induced modulations of the phospholipid signalling reactions do occur in vivo and that these effects are involved in the mechanism of beneficial effects of dietary PUFAs on the process of atherosclerosis.

Development and regression of atherosclerosis in pigs. Effects of n-3 fatty acids, their incorporation into plasma and aortic plaque lipids, and granulocyte function.

Fifty-one pigs were fed a low-cholesterol basal diet, to which either 10% (by weight) of lard fat (group INORM, n = 7), 2% cholesterol plus 8% lard fat (group II, n = 33), or 2% cholesterol plus 4% lard fat plus 4% fish oil (group IIIPREV, n = 11) was added. In all pigs, the left anterior descending coronary artery and the abdominal aorta were denuded at 1 month. In the first 24 hours thereafter, three animals in group II and two in group IIIPREV died suddenly. After 3 months, 0.5% bile acids was added to the diet in groups II and IIIPREV. After 8 months the degree of atherosclerosis was evaluated in groups INORM and IIIPREV and in 14 animals from group II (IIIND). At 4 months, one animal from Group II died of pneumonia. For the next 4 months (postinduction period), the remaining 15 animals from group II received the basal diet, to which either 10% lard fat (group IILF, n = 6) or 5% lard fat plus 5% fish oil (group IIFO, n = 9) was added. The hypercholesterolemic diet increased plasma cholesterol from 2 to 9-12 mM after 8 months. Fish oil had no major effects on plasma lipids during both induction and postinduction. Superoxide production by granulocytes in response to the membrane receptor-dependent N-formyl-methionyl-leucyl-phenylalanine (fMLP) gave a higher response in group IIIND than in group INORM. In group IIIPREV, the response to phorbol myristate acetate (PMA) and fMLP was lowered, while in groups IIFO and IILF the responses to PMA and fMLP were not affected. The response to serum-treated zymosan was similar in all groups. Abrasion caused increases in free cholesterol (40%) and phospholipids (46%) in the abdominal aortas of group INORM animals. Hypercholesterolemia increased both free and esterified cholesterol in the entire aorta. Fish oil prevented accumulation of free cholesterol in the nonabraded ascending aorta during induction and further accumulation of free cholesterol and phospholipids in the abdominal aorta during postinduction. In the nonabraded ascending aorta, triglycerides were significantly (almost five times) lower in group IIFO than in group IILF. During both induction and postinduction, a large incorporation of n-3 polyunsaturated fatty acids (up to 20%) occurred in plasma and aortic cholesterol esters and phospholipids of groups IIFO and IIIPREV.(ABSTRACT TRUNCATED AT 400 WORDS)

Myocardial phosphoinositides do not share the same fatty acid profile.

It is generally assumed that the fatty acid compositions of the phosphoinositides are identical. To investigate this in myocardium, inositol lipids extracted from rat and pig ventricular homogenates were absorbed to neomycin-coated glass beads, eluted and quantitated by fatty acid analysis after thin-layer chromatography. The percentages of stearic, oleic, linoleic and arachidonic acid (20:4n-6) in the rat were, respectively, 49, 4, 7 and 26 for phosphatidylinositol, 62, 1, 4 and 18 for phosphatidylinositol-4-monophosphate and 63, 2, 4, 18 for phosphatidylinositol-4,5-bisphosphate. Equal distribution patterns of fatty acids were found in homogenate and sarcoplasmic reticulum of pig myocardium. Cultured rat ventricular myocytes were utilized to study the incorporation (25 h) of [14C]20:4n-6 relative to that of myo-[3H]inositol into phosphatidylinositol and phosphatidylinositol-4,5-bisphosphate which were, respectively, 1.61 and 1.22. The data indicate that in myocardium phosphatidylinositol-4,5-bisphosphate represents a relatively modest source of 20:4n-6.

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.

Occurrence and functions of the phosphatidylinositol cycle in the myocardium.

In the last decade a great deal of attention was awarded to a signal transduction pathway which is utilized primarily by 'Ca2+ mobilizing' signal molecules and which involves the hydrolysis of a quantitatively minor phospholipid, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) by a PtdIns-specific phospholipase C (PLC). The evidence for the existence of receptor-mediated GTP binding protein-coupled PLC in myocardium and its possible functions are briefly summarized. The minireview is concentrated on the following aspects: 1) cellular localization and synthesis of polyphospho-PtdIns from PtdIns, 2) desensitization of the alpha 1-adrenergic agonist and endothelin-1 mediated PtdIns responses, 3) oscillatory Ca2+ transients initiated by PtdIns(4,5)P2 hydrolysis, 4) polyunsaturated fatty acids as constituents of polyphospho-PtdIns and of the protein kinase C activator 1,2-diacylglycerol (DAG), 5) source other than PtdIns(4,5)P2 contributing to the stimulated DAG, 6) role of the PtdIns pathway in cardiomyocyte growth and gene expression during the hypertrophic response.

Modification of fatty acid composition of the phospholipids of cultured rat ventricular myocytes and the rate of phosphatidylinositol-4,5-bisphosphate hydrolysis.

Cultured neonatal cardiac myocytes have been utilized as a model for the study of the role of fatty acids in the alpha 1-adrenoceptor mediated phosphatidylinositol turnover. Experiments were started 24 h after seeding, when there was a confluent monolayer of beating cardiomyocytes. The cells were incubated for 3-4 days in sera containing culture medium with (1) no additives or (2) a mixture of 107 microM 18:0 and 18:1n-9, or (3) only 214 microM 18:2n-6 or (4) 214 microM 20:5n-3. No differences in the cellular content of the various phospholipid classes among the different groups of fatty acid treated cells were found. The predicted elevations of 18:1n-9, 18:2n-6 and 20:5n-3 associated with a partial depletion of 20:4n-6 were confirmed in all phospholipid classes, except for sphingomyelin. The mol% of 18:0, 18:2n-6, 20:4n-6 and 20:5n-3 in the phosphatidylinositol fraction were respectively 39, 4, 30 and 0.6 for the control treated cells, 34, 3, 15 and 0 for 18:0/18:1n-9 treated cells, 40, 17, 24 and 0.2 for the 18:2n-6 treated cells and 41, 3, 13 and 21 for the 20:5n-3 treated cells. Apart from the observed reductions in the basal rates, the phenylephrine (30 microM) stimulated production of inositolphosphates was reduced by 51% and 71%, respectively in the 18:2n-6 and 20:5n-3 treated cardiomyocytes. The basal rate of inositolphosphate formation was 37% increased in the 18:0/18:1n-9 treated cells. The [3H]-inositol incorporation into phosphatidylinositol 4,5-bisphosphate was only slightly reduced by 18:2n-6 and 20:5n-3 treatments (respectively 12 and 28% compared to control treated cells). Prolonged (30 min) alpha 1-adrenergic stimulation did not affect the contents and fatty acid profiles of any class of phospholipid, not even phosphatidylinositol. In conclusion, variations in the polyunsaturated fatty acid composition of membrane phospholipids do affect the basal and the alpha 1-adrenoceptor stimulated rate of phosphatidylinositol-4,5-bisphosphate hydrolysis. The reducing effects of 18:2n-6 and 20:5n-3 treatment on the rate of inositolphosphate production may be partially ascribed to altered levels of phosphatidyl-inositol 4,5-bisphosphate.

Failure of diltiazem to suppress cholesterol-induced atherogenesis of endothelium-denudated arteries in pigs.

To investigate the effect of diltiazem on the development of atherosclerosis, 15 pigs were fed a fat-poor basal diet to which 8% (w/w) lard fat and 2% (w/w) cholesterol were added for 8 months. To enhance the formation of atherosclerotic plaques endothelium of the aorta and the left anterior descending coronary artery was removed after 1 month and 0.5% (w/w) bile acids were added to the diet after 3 months. Seven animals served as control, while 8 animals also received diltiazem (the first 2 months 10 mg/kg twice daily and during the remainder of the dietary period 5 mg/kg twice daily). The diet-induced increases in plasma level of total cholesterol were not affected by diltiazem. Triglyceride levels did not change in the control group but decreased significantly in the diltiazem-treated animals. Collagen-induced (1 microgram/ml) platelet aggregation was increased by diltiazem. The sum of free and esterified cholesterol was increased in the lesions of the aortic wall in the diltiazem-treated animals (9.8 +/- 1.3 micrograms/g wet weight vs. 6.3 +/- 1.0 mumol/g wet weight in the untreated animals), but coverage of the aorta with sudanophilic lesions was similar for both groups (40 +/- 4% for the treated and 34 +/- 9% for the control animals). Narrowing of the previously abraded coronary arteries was similar for the diltiazem-treated (median 7.1%, ranges 2.6-29.0%) and the control group (median 10.0%, ranges 2.3-24.1%). It is concluded that the dose range of diltiazem of 5-10 mg/kg twice daily, which is close to that used in the clinical setting, had no effect on the experimentally induced atherogenesis in pigs.

Alterations in polyunsaturated fatty acid composition of cardiac membrane phospholipids and alpha 1 adrenoceptor mediated phosphatidylinositol turnover.

STUDY OBJECTIVE - The aim of the study was to investigate the steps at which polyunsaturated fatty acids are involved in alpha 1 adrenoceptor mediated phosphatidylinositol turnover. DESIGN - Phosphatidylinositol turnover rates were investigated after preincubating neonatal rat ventricular myocytes with culture media enriched with linoleic acid (18:2n-6) or eicosapentaenoic acid (20:5n-3) to change the polyunsaturated fatty acid composition of their membrane phospholipids. EXPERIMENTAL MATERIAL - Cardiomyocytes were isolated from ventricles of 2-4 d old Wistar rats by trypsinization and were then cultured. Experiments were started 48 h after seeding, when there was a confluent monolayer of beating cardiomyocytes. MEASUREMENTS and RESULTS - In 18:2n-6 treated cells the 18:2n-6 content in the total phospholipid fraction rose from 45 to 68 nmol.mg-1 protein; in 20:5n-6 treated cells the 20:5n-3 content rose from 1.5 to 12.5 nmol.mg-1 protein, and the docosapentaenoic acid (22:5n-3) content rose from 5.1 to 14.7 nmol.mg-1 protein. The major n-3 fatty acid, 22:6n-3 (11.4 nmol.mg-1 protein), did not change after 20:5n-3 treatment. Although the phosphatidylinositol fraction showed changes paralleling those in the total phospholipids, none were significant. In this fraction the major n-3 fatty acid appeared to be 22:5n-3 (0.4 nmol.mg-1 protein). The fatty acid treated cells were prelabelled with [3H]-inositol to estimate the rate of phosphatidylinositol-4,5-bisphosphate turnover. There were no differences in the rate of [3H]-inositolphosphate formation between control, 18:2n-6 treated cells, and 20:5n-3 treated cells. Prolonged alpha 1 adrenergic stimulation of control and treated cells did not change the polyunsaturated fatty acid composition of the total phospholipid and phosphatidylinositol fractions. CONCLUSIONS - The alpha 1 adrenoceptor mediated phosphatidylinositol turnover rate is not affected by changes in polyunsaturated fatty acid composition of membrane phospholipids, neither does prolonged alpha 1 adrenergic stimulation lead to significant depletion of any specific or total polyunsaturated fatty acids in the phosphatidylinositol lipids.