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 α1-adrenoceptor mediated phosphatidylinositol turnover. Experiments were started 24h 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 μm 18:0 and 18:1n-9, or (3) only 214 μm 18:2n-6 or (4) 214 μm 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 (30μm) 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) α1-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 α1-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 phosphatidylinositol 4,5-bisphosphate.</p

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 α1-adrenoceptor mediated phosphatidylinositol turnover. Experiments were started 24h 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 μm 18:0 and 18:1n-9, or (3) only 214 μm 18:2n-6 or (4) 214 μm 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 (30μm) 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) α1-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 α1-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 phosphatidylinositol 4,5-bisphosphate

Alterations in polyunsaturated fatty acid composition of cardiac membrane phospholipids and α₁ adrenoceptor mediated phosphatidylinositol turnover

Study objective - The aim of the study was to investigate the steps at which polyunsaturated fatty acids are involved in α1 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-1protein. 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 α1 adrenergic stimulation of control and treated cells did not change the polyunsaturated fatty acid composition of the total phospholipid and phosphatidylinositol fractions. Conclusions - The α1 adrenoceptor mediated phosphatidylinositol turnover rate is not affected by changes in polyunsaturated fatty acid composition of membrane phospholipids, neither does prolonged α1 adrenergic stimulation lead to significant depletion of any specific or total polyunsaturated fatty acids in the phosphatidylinositol lipids.</p

Alterations in polyunsaturated fatty acid composition of cardiac membrane phospholipids and α1 adrenoceptor mediated phosphatidylinositol turnover

Study objective - The aim of the study was to investigate the steps at which polyunsaturated fatty acids are involved in α1 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-1protein. 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 α1 adrenergic stimulation of control and treated cells did not change the polyunsaturated fatty acid composition of the total phospholipid and phosphatidylinositol fractions. Conclusions - The α1 adrenoceptor mediated phosphatidylinositol turnover rate is not affected by changes in polyunsaturated fatty acid composition of membrane phospholipids, neither does prolonged α1 adrenergic stimulation lead to significant depletion of any specific or total polyunsaturated fatty acids in the phosphatidylinositol lipids

Excess FGF-7 in Corneal Epithelium Causes Corneal Intraepithelial Neoplasia in Young Mice and Epithelium Hyperplasia in Adult Mice

We hypothesized that human ocular surface squamous neoplasia (OSSN) may result from the continuous growth stimulation of corneal epithelial progenitor cells. In the present study, we analyzed the effects of excess fibroblast growth factor-7 (FGF-7) on both the proliferation and differentiation of corneal epithelium in a novel Krt12-rtTA/tet-O-FGF-7 double transgenic mouse model in which cornea-specific FGF-7 overexpression is achieved by doxycycline (Dox) treatment. When such adult mice were exposed to Dox, they exhibited epithelial hyperplasia with increases in phospho-extracellular signal-regulated kinase 1/2-, nuclear β-catenin-, and 5-bromo-2′-deoxyuridine-labeled cells and altered keratin (K) 14 (K14) expression pattern, a normal K12 expression pattern, and the normal absence of K10. Hyperplasia of the adult cornea was fully reversible 2 weeks after the removal of Dox from chow. In contrast, double transgenic embryos that were exposed to Dox from embryonic day 0.5 to postnatal day 21 developed papillomatous tumors in the cornea, resembling human OSSN, and ectopic gland-like structures in the limbus, accompanied by the down-regulation of K12 and the up-regulation of K14, Pax6, and p63. These epithelial anomalies observed in young experimental mice were not fully resolved after the termination of Dox induction. Taken together, Krt12-rtTA/tet-O-FGF-7 mice may be a suitable animal model for the study of the molecular and cellular mechanisms of human OSSN

First-line treatment with infliximab versus conventional treatment in children with newly diagnosed moderate-to-severe Crohn's disease : An open-label multicentre randomised controlled trial

Objective: In newly diagnosed paediatric patients with moderate-to-severe Crohn's disease (CD), infliximab (IFX) is initiated once exclusive enteral nutrition (EEN), corticosteroid and immunomodulator therapies have failed. We aimed to investigate whether starting first-line IFX (FL-IFX) is more effective to achieve and maintain remission than conventional treatment. Design: In this multicentre open-label randomised controlled trial, untreated patients with a new diagnosis of CD (3-17 years old, weighted Paediatric CD Activity Index score (wPCDAI) >40) were assigned to groups that received five infusions of 5 mg/kg IFX at weeks 0, 2, 6, 14 and 22 (FL-IFX), or EEN or oral prednisolone (1 mg/kg, maximum 40 mg) (conventional). The primary outcome was clinical remission on azathioprine, defined as a wPCDAI <12.5 at week 52, without need for treatment escalation, using intention-to-treat analysis. Results: 100 patients were included, 50 in the FL-IFX group and 50 in the conventional group. Four patients did not receive treatment as per protocol. At week 10, a higher proportion of patients in the FL-IFX group than in the conventional group achieved clinical (59% vs 34%, respectively, p=0.021) and endoscopic remission (59% vs 17%, respectively, p=0.001). At week 52, the proportion of patients in clinical remission was not significantly different (p=0.421). However, 19/46 (41%) patients in the FL-IFX group were in clinical remission on azathioprine monotherapy without need for treatment escalation vs 7/48 (15%) in the conventional group (p=0.004). Conclusions: FL-IFX was superior to conventional treatment in achieving short-term clinical and endoscopic remission, and had greater likelihood of maintaining clinical remission at week 52 on azathioprine monotherapy. Trial registration number: ClinicalTrials.gov Registry (NCT02517684).publishedVersionPeer reviewe